Therapeutic methods and compositions

ABSTRACT

Methods for diagnosing or treating immune disorders in a subject are provided. The methods are based on the detection or modulation of Refractory state Inducing Factor (RIF).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/105,042, filed Jun. 16, 2016, now U.S. Pat. No. 11,365,264, which isthe U.S. national stage application of International Patent ApplicationNo. PCT/EP2014/078969, filed Dec. 22, 2014, which claims the benefit ofU.S. Provisional Patent Application No. 61/920,137, filed Dec. 23, 2013and U.S. Provisional Patent Application No. 62/017,457, filed Jun. 26,2014.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Jun. 14, 2016 and is 33 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for modulatingthe immune system in a subject in need thereof. The invention moreparticularly discloses the existence and characterization of a keyendogenous factor of the immune response and provides novel therapeuticand diagnostic methods and compositions based on a modulation of thisfactor. The invention particularly provides compositions and methodssuitable to stimulate or inhibit CD4 T cell-mediated immune responses ina subject, as well as methods and compositions for monitoringimmunodeficiency, including immunodeficiency associated with humanimmunodeficiency virus (HIV) infection. Also provided are methods andcompositions to diagnose and assay CD4 T-cell-defects that persist afterantiretroviral therapy, as well as methods to develop drugs able tospecifically treat this immunodeficiency.

INTRODUCTION

CD4 T lymphocytes play a pre-eminent role in controlling the immunesystem (both cellular and humoral responses) and are critical in variousdisease conditions.

During the immunological disease associated with HIV pathogenesis, lessthan 0.5% of all CD4 T cells are actually infected (as measured in theperipheral blood), but the great majority of CD4 T cells shows majorregulatory dysfunction. Uninfected CD4 T lymphocytes progressively losetheir function, become anergic, and their numbers decrease resulting inCD4 lymphopenia. Anergy and lymphopenia are the hallmarks of theimmunodeficiency characterizing HIV-infected patients. The mechanismsbehind these phenomena have never been fully elucidated (1).

Immune activation and inflammation also play a critical role in HIVpathogenesis (2, 3). The inventors have previously demonstrated that adecrease in responsiveness to interleukin-2 (IL-2), leading to CD4anergy (4), and a reduction in responsiveness to interleukin-7 (IL-7)which, by disrupting the IL-7/CD4 regulatory loop, participates in themechanisms leading to CD4 lymphopenia (5). The mechanisms involved havebeen attributed to defects in the Janus kinase (Jak)/Signal Transducerand Activator of Transcription (STAT) pathway (6, 7). Similar resultshave been obtained by other laboratories (8, 9). In this regard,compartmentalization of the IL-7 receptor (IL-7R) is required toinitiate normal CD4 T cell responses (10). Upon IL-7 binding, the twochains of the IL-7R (IL-7R alpha and gamma-c) are first driven intomembrane microdomains (MMD). These are cellular compartments which, likelipid rafts, are rich in cholesterol and sphingomyelin, but they alsocontain very significant amounts of structural and functional proteins(11). IL-7R complexes induce a reorganization of the cytoskeleton whichthen interacts with its meshwork. These two successive steps would berequired for initiation of the Jak/STAT pathway (12).

The present inventors have investigated the mechanisms behind theunresponsiveness of CD4 T lymphocytes in viremic HIV-infected patients(VP). The experiments provided herein demonstrate that chronicactivation of CD4 T lymphocytes drives them into an aberrant state ofactivation/differentiation which renders them refractory to certainphysiological signals such as those delivered by interleukin-7.Furthermore, the present invention reports the identification, isolationand characterization, from human plasma, of the protein responsible forthis aberrant state of CD4 T cell activation. For the first time, theinvention thus discloses that immunosuppression can be mediated by anendogenous circulating protein which, upon expression, is able to inducealteration and inactivation of CD4-T cells and, upon inhibition, canstimulate the immune system in a subject.

Based in part on these remarkable findings, the invention now providesnew methods, compositions and compounds for modulating the immunesystem, particularly for modulating the immune system in subjects havingaltered immunity (e.g.; immuno-depressed or pathologic immunereactions). The invention further provides novel methods for treatingimmune disorders by modulating CD4 T cells. The invention isparticularly suited for treating immunodeficiencies linked to CD4 T cellimpairment, such as immunodeficiency syndrome associated withHIV-infection. The invention also provides reagents and methods forcharacterizing the aberrant activation state, reactiveness to IL7 and/orfor monitoring immunoresponse impaired in HIV infected patients.Response of CD4 T cells can be evaluated in untreated or treatedpatients with antiretroviral drugs, and qualify their response totreatment and evaluate the competency of their CD4 T cells.

SUMMARY OF THE INVENTION

An object of the invention relates to a method for modulating an immuneresponse in a subject, comprising exposing the subject to a compoundthat modulates the amount (e.g., expression) or activity of GIBsPLA2.

A further object of the invention relates to a method of treatment of animmune disorder in a subject, comprising exposing the subject to acompound that modulates the amount (e.g., expression) or activity ofGIBsPLA2.

A further object of the invention relates to a method of treatment of animmune disorder in a subject, comprising modulating the amount (e.g.,expression) or activity of GIBsPLA2 in the subject.

Another object of the invention relates to the use of a compound thatmodulates the amount (e.g., expression) or activity of GIBsPLA2 for themanufacture of a medicament for modulating an immune response or fortreating an immune disorder in a subject.

Another object of the invention relates to a GIBsPLA2 modulator for usein a method of modulating an immune response or of treating an immunedisorder in a subject.

Another object of the invention relates to a GIBsPLA2 modulator for useto modulate white blood cells in a subject.

In a first embodiment, the invention is used to induce or stimulate animmune response in the subject, and comprises inhibiting GIBsPLA2 insaid subject, or exposing the subject to a GIBsPLA2 inhibitor. Suchembodiment is particularly suited to treat immuno-deficient subjects orsubject in need of stimulated immunity (e.g., infectious diseases,cancer, etc.).

A particular object of the invention thus resides in a method ofstimulating an immune response in a subject, comprising inhibitingGIBsPLA2 in said subject or exposing the subject to a GIBsPLA2inhibitor.

A further object of the invention relates to a method of treating aninfectious disease in a subject, comprising inhibiting GIBsPLA2 in saidsubject or exposing the subject to a GIBsPLA2 inhibitor.

A more particular embodiment of the invention relates to a method oftreating AIDS in a HIV-infected subject, comprising inhibiting GIBsPLA2in said subject or exposing the subject to a GIBsPLA2 inhibitor.

In a particular embodiment, exposing the subject to an inhibitorcomprises administering the inhibitor to the subject. In anotherembodiment, exposing the subject to an inhibitor comprises vaccinatingthe subject against GIBsPLA2.

In this regard, in a particular embodiment, the invention relates to amethod for stimulating the immune system of a subject in need thereof,the method comprising vaccinating the subject against GIBsPLA2.

In another particular embodiment, the invention relates to a GIBsPLA2antigen for use to vaccinate a subject in need thereof.

In another aspect, the invention is used to reduce or suppress anunwanted or deleterious immune response in the subject, and comprisescausing or increasing GIBsPLA2 in said subject, or exposing the subjectto a GIBsPLA2 agonist or activator. Such embodiment is particularlysuited to treat subjects having abnormal and/or pathologic immuneresponses (e.g., auto-immune diseases, inflammation, urticaria, eczema,allergies, asthma, etc.).

In a further aspect, the invention provides methods for diagnosing humanimmunodeficiency associated with CD4 T cell alteration. In someembodiments the methods comprise (a) providing a sample containing abody fluid, preferably plasma from a subject, and (b) detecting thepresence of GIBsPLA2 in the sample. In some embodiments of the methodsthe immunodeficiency is immunodeficiency associated with humanimmunodeficiency virus (HIV) infection. In some embodiments the methodcomprises contacting the sample with an antibody specific for GIBsPLA2.In some embodiments of the methods the presence of GIBsPLA2 in thesample is detected by an enzyme-linked immunosorbent assay (ELISA).

In another aspect, the invention provides methods for identifyingcandidate immunodeficiency therapeutic agents. In some embodiments theimmunodeficiency is associated with CD4 T cell alteration. In someembodiments of the methods, the human immunodeficiency associated withCD4 T cell alteration is caused by viral infection, particularly humanimmunodeficiency virus (HIV) infection. In some embodiments the methodscomprise: (a) contacting CD4 T lymphocytes with GIBsPLA2 in the presenceof an agent, (b) measuring GIBsPLA2-induced CD4 T cell activation, and(c) comparing the level of GIBsPLA2-induced CD4 T cell activation in thepresence of the agent with the level of GIBsPLA2-induced CD4 T cellactivation in the absence of the agent. In some embodiments of themethods, if the level of GIBsPLA2-induced CD4 T cell activation in thepresence of the agent is lower than the level of GIBsPLA2-induced CD4 Tcell activation in the absence of the agent, then the agent isidentified as a candidate immunodeficiency therapeutic agent. In someembodiments of the methods, if the level of GIBsPLA2-induced CD4 T cellactivation in the presence of the agent is not lower than the level ofGIBsPLA2-induced CD4 T cell activation in the absence of the agent, thenthe agent is identified as a candidate immunosuppressing therapeuticagent. In some embodiments the methods comprise measuringGIBsPLA2-induced CD4 T cell activation by determining the number of MMDper CD4 T cell. In some embodiments the methods comprise measuringGIBsPLA2-induced CD4 T cell activation by determining the mean diameterof MMD on CD4 T cells. In some embodiments the methods comprisemeasuring GIBsPLA2-induced CD4 T cell activation by determining the IL-7responsiveness of CD4 T cells.

In another aspect, the invention relates to a pharmaceutical compositioncomprising a GIBsPLA2 modulator and a pharmaceutically acceptablecarrier or excipient. In a preferred embodiment, the GIBsPLA2 modulatoris a GIBsPLA2 inhibitor, more preferably selected from an antibody or afragment or derivative thereof, an inhibitory nucleic acid, a peptide ora small drug. In another particular embodiment, the GIBsPLA2 modulatoris a GIBsPLA2 agonist or activator, more particularly a GIBsPLA2protein.

In another aspect, the invention relates to a vaccine compositioncomprising a GIBsPLA2 antigen (e.g., an immunogenic GIBsPLA2 protein oran epitope-containing fragment thereof), a pharmaceutically acceptablecarrier or excipient and, optionally, an adjuvant. In a preferredembodiment, the GIBsPLA2 antigen is a GIBsPLA2 protein or a fragmentthereof treated to (i) increase its immunogenicity in human subjectsand/or to (ii) reduce its biological activity.

The invention may be used in any mammal. It is particularly suited foruse in human subjects. It may be used to increase the immune response inany mammal, and it is particularly adapted to induce potent CD4-T cellactivity in immuno-depressed subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show that, before any stimulation, CD4 T cells from VP showan aberrant state of activation with many large membrane microdomainsthat are unaffected by IL-7.

FIG. 1A: Membrane microdomains (MMD) were labelled with cholera toxinsubunit B (CtxB-AF488) and analyzed by STED microscopy. From top tobottom, purified CD4 T cells from HD, VP and PHA-activated (40 μg/ml, 30min) HD T cells. For each group the top half of a representative CD4T-cell before and after IL-7 stimulation (2 nM, 15 min) is shown fromZ-stack image series. CD4 T lymphocytes were also treated withcholesterol oxidase (COase, 31 μM, 25 min) plus sphyngomyelinase (SMase,2.7 μM, 5 min) before stimulation by IL-7.

FIGS. 1B-1C: MMD were counted on the entire surface of the purified CD4T cells. An average of 50 cells were examined. (FIG. 1B) HD cells before(HDc: NS) and after IL-7 stimulation (HDc: IL-7). (FIG. 1C) VP cellsbefore (VPc: NS) and after IL-7 stimulation VP (VPc: IL-7),PHA-activated HD cells before (HDc: PHA) and after IL-7 stimulation(HDc: PHA/IL-7).

FIGS. 1D-1E: MMD size was measured at the surface of purified CD4 Tcells (FIG. 1D) IL-7-stimulated HD cells (HDc: IL-7), (FIG. 1E)IL-7-stimulated VP cells (VPc: IL-7) and IL-7-stimulatedPHA-pre-activated HD cells (HDc: IL-7).

FIGS. 2A-2C show that IL-7R chains from VP CD4 T-cells are embedded indetergent-resistant microdomains (DRM) that are unaffected by IL-7.Purified CD4 T lymphocytes were lysed (0.5% Triton X-100) and 200 μl ofthe lysate was loaded on a 5-40% sucrose gradient. After 16 h ofcentrifugation (50 krpm) at 4° C., 18 fractions were collected (#1left=tube top=5% sucrose; #18 right=tube bottom=40% sucrose). Eachfraction was analyzed on SDS-PAGE (7% acrylamide-bis). Flottilin, IL-7Ralpha and gamma-c were detected by immunoblotting (10).

FIG. 2A: Flottilin was used as a marker to indicate low densityfractions corresponding to DRM and high-density fractions outside rafts.

FIG. 2B: IL-7Ralpha and (FIG. 2C) gamma-c bands are shown for purifiednon-stimulated HD CD4 T-cells (HDc: NS), IL-7-stimulated HD cells(HDc:IL-7), non stimulated VP cells (VPc:NS) and PHA-activated HD cells(HDc:PHA).

FIGS. 3A-3E show that IL-7R function is altered in membrane microdomainsof VP CD4 T-cells.

FIG. 3A: Two-dimensional effective diffusion rates D_(eff) forIL-7Ralpha were measured as developed in FIG. 7 . Diffusion rates werealso measured after adding various drugs: COase (31 μM, 30 min) plusSMase (2.7 μM, 5 min) (CO/SM), Col (10 μM, 30 min) plus CytD (20 μM, 30min) (CytD/Col), or in the presence of all these inhibitors (all). CD4 Tcells from HD (HDc) and VP (VPc) were studied, as were PHA-activated HDCD4 T cells (HDc: PHA). Bars indicate SEM from 5 independentexperiments. More experimental data are given in FIG. 8 .

FIG. 3B: IL-7-induced phosphorylation and nuclear translocation of STATSwere followed using rabbit phospho-STATS labelled with goatanti-rabbit-Atto642 and analyzed by pulsed-STED microscopy (0.5 μmslices). The experiments involved purified non stimulated HD CD4 T cells(HDc: NS), IL-7-stimulated HD CD4 T cells (HDc: IL-7), non stimulated VPCD4 T cells (VPc: NS), IL-7-stimulated VP CD4 T cells (VPc:IL-7),PHA-activated HD CD4 T cells (HDc:PHA) and PHA-activated HD CD4 T cellsstimulated by IL-7 (HDc:PHA/IL-7). The effects of colchicine pluscytochalasin D are shown in the left panel.

FIGS. 3C-3E: After IL-7 stimulation, the kinetics of phospho-STATSappearance in the cytoplasm and accumulation in the nucleus weremeasured using ImageJ software. (FIG. 3C) HD CD4 T cells (black line)and HD CD4 T cells treated with Col plus CytD (grey line), (FIG. 3D) VPCD4 T cells (black line) and (FIG. 3E) PHA-activated HD CD4 T cells(grey line).

FIGS. 4A-4D show that plasma from VP induces an aberrant activationpattern in HD CD4 T cells as measured by the number of MMD.

FIG. 4A: Representative images of HD CD4 T cells treated with plasma(10%) from VP (HDc: VPp), HIC (HDc: HICp) or ART patients (HDc: ARTp)are shown. MMD were stained with cholera toxin (CtxB-AF488). For eachgroup the top half of a representative CD4 T-cell from Z-stack imagesbefore (left) and after IL-7 stimulation (2 nM, 15 min) (right) isshown.

FIG. 4B: MMD induced at the surface of CD4 T-cells (HDc) by plasmas(10%) from 5 different VP (VPp1 to VPp5). Results were obtained from theanalysis of 50 cells before (white) and after (gray) IL-7 stimulation.Mean values and quartiles are shown.

FIG. 4C: Comparison of the effects of plasmas from HD (HDp), VP (VPp),HIC (HICp) and ART patients (ARTp) after (gray) and before (white) IL-7stimulation.

FIG. 4D: Dose (0.01% to 10%)-response obtained with the plasmasdescribed in c. The number of MMD induced at the surface of HDc CD4T-cells is shown. The effect of VP plasma is shown as a plain line.

FIGS. 5A-5D show that plasma from VP inhibits IL-7-induced STATSphosphorylation and nuclear translocation of phospho-STATS in HD CD4 Tlymphocytes.

FIG. 5A: Before IL-7 stimulation, purified HD CD4 T cells werepre-incubated with plasma (10%). IL-7-induced phosphorylation andnuclear translocation of phospho-STATS were followed by pulsed-STEDmicroscopy (0.5 μm slice). The following plasmas (10%) were studied:control (HDc: NS), VP (HDc: VPp), HIC (HDc: HICp) and ART patients (HDc:ARTp).

FIG. 5B: Analysis of phospho-STATS recovered in the cytoplasm (white)and nucleus (gray) of IL-7-stimulated HD CD4 T-cells pre-treated withplasmas from 5 different VP (10%).

FIG. 5C: Comparison of the effects of plasma (10%) pre-incubation onIL-7-stimulated HD CD4 T cells. Plasma were from HD (HDp), VP (VPp), HIC(HICp) and ART patients (ARTp).

FIG. 5D: Dose (0.01%-10%)-response obtained with the plasmas as measuredby the inhibition of phospho-STATS nuclear translocation inIL-7-stimulated HD CD4 T-cells. The effect of VP plasma is shown as aplain line.

FIGS. 6A-6D show molecular characterization of the Refractory stateInducing Factor (RIF) recovered from VP plasma.

FIG. 6A: Treatment of VP plasma by trypsin, DNase, RNase and PNGase. RIFactivity was followed by measuring the number of MMD and effects onIL-7-induced nuclear phospho-STATS in HD CD4 T-cells.

FIG. 6B: RIF MW was measured by gel filtration on a Sephadex G100column. RIF activity on HD CD4 T-cells was followed by measuring thenumbers of MMD induced by the different fractions of the column. Eachfraction was also tested for the presence of viral proteins by dot blotusing polyclonal antibodies from VP plasma. Background obtained with HDplasma has been subtracted. Experiments were repeated three times.

FIG. 6C: RIF MW was also measured after gel filtration on a SephadexG100 column and its activity followed by inhibition of IL-7-inducedphospho-STATS as measured by FACS. Percentages of maximum IL-7-inducedphospho-STATS were recorded. The amount of protein in each fraction isalso reported. Experiments were repeated twice.

FIG. 6D: Isoelectric point was measured as follows. RIF eluted from theSephadex G100 column was loaded onto an anion (MonoQ) or cation (MonoS)exchange column. RIF activity was eluted by pH-step buffers. The numberof MMD on HD CD4 T-cells was plotted against pH.

FIGS. 7A-7C show a 2D gel analysis of the IL-7 signalosome in purifiedCD4 T cells from HD, VP and IL-7-stimulated HD cells. (FIG. 7A)non-stimulated (NS) HD CD4 T-cells. (FIG. 7B) VP CD4 T-cells. (FIG. 7C)IL-7-stimulated HD CD4 T-cells.

FIGS. 8A-8G show an analysis of the diffusion rate of IL-7Ralpha at thesurface of purified CD4 T cells from HD, VP and PHA-stimulated HD cells.(FIGS. 8A, 8D) at the surface of HD CD4 T-cells, (FIGS. 8B, 8E) at thesurface of VP CD4 T cells, (FIGS. 8C, 8F) at the surface of HD CD4 Tcells pre-activated with PHA (1 μg/ml). (FIG. 8G) Scheme of themechanism of IL-7Ralpha diffusion embedded in MMD before and aftertreatment by MMD inhibitors or cytoskeleton inhibitors.

FIGS. 9A-9D show a schematic representation of the hypothetical mode ofaction of RIF on HD CD4 T cells and mechanism of IL-7 unresponsiveness.RIF induces abnormal MMD which are non functional. The IL-7 signalosomeis therefore altered and the cells remain unresponsive to the cytokine,as in VP CD4 T cells. Aberrant activation patterns and signallingdefects in RIF-induced HD CD4 T cells and in VP CD4 T cells areundistinguishable. The left part of the scheme illustrates the differentsteps in the mechanisms of IL-7 signal transduction in HD (10, 12).

FIG. 9A: In resting CD4 T cells, before IL-7 recognition, the IL-7Rchains are associated but their intracytoplasmic domains are far apartand the signaling molecules Jak1 and Jak3 are not interacting.

FIG. 9B: In IL-7-activated CD4 T cells, the IL-7R is compartmentalizedin normal MMD (90 nm in diameter) and the signalosome becomesfunctional. After cytoskeleton organization, STATSA and STATSB arephosphorylated in contact with the IL-7R/Jak1/Jak3 complexes thenmigrate to the nucleus by moving along the microtubules as previouslydiscussed (12).

The right part of the scheme illustrates the hypothetical mechanism ofaction of RIF. The proposed mechanism of action is derived frompreliminary data and comparison of RIF-induced defects with thealterations characterized in purified CD4 T cells from VP (unpublisheddata).

FIG. 9C: RIF induces many large abnormal MMDs. IL-7Rs are embedded inabnormal MMDs and their ability to induce a functional signalosome isaltered.

FIG. 9D: RIF-treated HD CD4 T cells are unresponsive to IL-7. Jak1 andJak3 phosphorylate STATS, although with reduced kinetics, butphospho-STATS do not migrate into the nucleus due to the lack ofcytoskeleton and microtubules organization.

FIGS. 9A-9D show STED microscopy images of MMD labelled with CtxB: AF488(half pile of Z-stack from CW-STED). FIGS. 9B and 9D show tubulinstained with rabbit anti-tubulin/goat anti-rabbit-Atto642, actin stainedwith mouse anti-actin/goat-anti-mouse-Chr494 and phospho-STATS stainedwith rabbit anti-phospho-STATS/goat-anti-rabbit-Atto642. Pulsed-STEDmicroscopy shows a 0.5 μm slice of methanol-permeabilized CD4 T-cells.After IL-7 stimulation, actin in the MMD cytoplasmic area of RIF-treatedHD CD4 T lymphocytes fails to concentrate as structured pads and doesnot form a cortex surrounding the nucleus, unlike in HD. Furthermore,the tubulin in these RIF-treated HD CD4 T cells, like in VP CD4 T cells,fails to form microtubules which have been hypothesized as beingcritical rods bridging the cytoplasm and nuclear membrane and therebyessential for STATS nuclear translocation.

Summary of the defects: Circled numbers 1, 2, 3 and 4 indicate thedifferent defective steps related to the aberrant activation pattern andIL-7 unresponsiveness in RIF-treated HD T cells: (1) abnormal proteinpattern of signalling complexes as described by 2D-gels, (2) abnormalmembrane structures such as large MMD as seen by STED microscopy, (3)abnormal cytoskeleton organization as measured by diffusion kinetics andSTED microscopy, and (4) abnormal signalling intermediate and inhibitionof phospho-STATS nuclear translocation as shown by STED microscopy.

FIGS. 10A-10B: PLA2sGIB inhibits IL-2 induced PStat5 nucleartranslocation in CD4 T cells of healthy donors (HD): Resting CD4 T cellspurified from 4 healthy donors were treated for 30 minutes at 37° C.with 3% or 1% of plasma from 5 VP (VP63, VP68, VP69, VP74 and VP75) andfrom 3 HD used as control. When indicated they were stimulated with 2 nMIL-2 for 15 minutes at 37° C. The percentage of cells positive fornuclear PStat5, with mean and SD, in whole CD4 T cells (FIG. 10A) and inCD4+ CD25+ T cells (FIG. 10B), before and after IL-2 stimulation areshown. Intracellular localisation of PStat5 was observed using LaserScanning Confocal Microscopy (LSM 700, Zeiss) after indirect stainingwith rabbit anti human PStat5 (pY694) followed by donkey anti rabbitIgG-Die light 405. Total CD4 T cells were stained with goat anti humanb-Tubulin followed by donkey anti goat IgG-AF555. CD25+ CD4 T cells weretargeted with a mouse anti human CD25 followed by donkey anti mouseIgG-AF488.

FIG. 11 : PLA2sGIB inhibits IL-4 induced PStat6 nuclear translocation inCD4 T cells of healthy donors (HD): Resting CD4 T cells purified from 4healthy donors were treated for 30 minutes at 37° C. with 3% or 1% ofplasma from 5 VP (VP63, VP68, VP69, VP74 and VP75) and from 3 HD used ascontrol. When indicated they were stimulated with 2 nM IL-4 for 15minutes at 37° C. The percentage of cells positive for nuclear PStat6,with mean and SD, in whole CD4 T cells, before and after IL-2stimulation are shown. Intracellular localisation of PStat6 was observedusing Laser Scanning Confocal Microscopy (LSM 700, Zeiss) after indirectstaining with rabbit anti human PStat6 (pY694) followed by goat antirabbit IgG-AF488. Total CD4 T cells were stained with mouse anti humana-Tubulin followed by goat anti mouse IgG-AF647.

FIG. 12 : Absence of activity of mutant pPLA2GIB H48Q.

FIGS. 13A-13B: Comparison of the activity of wild type cloned porcinePLA2 GIB and of its mutant H48Q. FIG. 13A: induction of abnormalMembrane Microdomains (aMMD); FIG. 13B: effect on the IL-7 inducedNuclear Translocation of phosphoSTAT5 (NT of pSTAT5).

FIGS. 14A-14B show the treatment of plasma from viremic patients withgoat anti-PLA2 GIB antibodies coupled to sepharose beads. Aftertreatment (30 min at room temperature) the plasmas were tested:

FIG. 14A: The percentage of CD4 T cells showing abnormal MMD/cell wasmeasured after staining with Cholera toxin B (CtxB-AF488).

FIG. 14B: The nuclear translocation of pSTAT5 was measured after IL-7stimulation and the percentage of positive nucleus counted.

FIG. 15 : Effect of anti-PLA2 GIB antibodies on the induction of aMMDand inhibition of NT pSTAT5.

FIG. 16 : Soluble PLA2GIB mouse receptor (sMR) inhibits the activity ofhuman PLA2GIB (huPLA2GIB) on the response to IL-7 of CD4 T cells fromhealthy donors, expressed as the percent of cells positive for nucleartranslocation of PStat5. The restoration of the response is calculatedas:

100×(% Pos cell_(huG1B+sMR)−% Pos cell_(huG1B))/(% Poscell_(culture medium)−% Pos cell_(huG1B))

FIGS. 17A-17B show the plasma from CD4 non-responder (CD4-NR) patientsinduce aberrant MMD in HD CD4 T cells—(FIG. 17A) Images of HD CD4 Tcells treated with plasma (1%) from CD4-NR patient obtained usingStructured Illumination Microscopy (SIM). MMD were stained with choleratoxin B (CtxB-AF488). Projection of Z-stacks images of a representativeCD4 T cell is shown. After IL-7 stimulation (2 nM, 15 min) there is nomodification of the image (right). (FIG. 17B) Dose curve response(0.0001% to 1%) obtained with plasmas from 5 CD4-NR patients and from arepresentative viremic patient. The number of abnormal MMD induced atthe surface of HD CD4 T cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for modulatingthe immune system in a subject in need thereof. The invention moreparticularly discloses the identification of GIBsPLA2 as a keyendogenous factor of the immune response and provides novel therapeuticand diagnostic methods and compositions based on a modulation of thisfactor.

A hypothesis of the present invention was that chronic activation of theimmune system in HIV-infected patients is abnormal and drives CD4 Tcells into an aberrant state of activation/differentiation that isunresponsive to the gamma-c cytokines involved in controlling manyaspects of immune defenses and homeostasis of the CD4 compartment,despite the fact that more than 99.5% of CD4 T cells from the peripheralcompartment are uninfected. This hypothesis was evaluated by theinventors and the present invention ellucidates the nature andsignificance of this aberrant state of activation.

More specifically, in a first aspect, the present invention demonstratesthat the characteristics of this state may be summarized as follows: 1)before any stimulation, all the CD4 T cells in Viremic HIV-infectedpatients (VP) possess numerous large MMD on their surface, 2) theseabnormal MMD sequester all the cell's IL-7Ralpha and gamma-c chains and3) this sequestering of the chains in abnormal MMD alters their abilityto induce the formation of a functional signalosome, 4) leading to aslowdown and a reduction of STATS phosphorylation and 5) a reduction ofphospho-STATS nuclear import. This abnormal pattern of pre-existing MMDon the surface of VP CD4 T lymphocytes has multiple consequences and isa basic mechanism explaining the various manifestations of theimmunodeficiency in HIV-infected patients. Loss of IL-7 responsivenessis an important factor that partly explains the CD4 lymphopeniaobserved. The persistent loss of these cells in VP—due to theirsensitivity to apoptosis and their destruction by low-level butcontinuous virus proliferation—cannot be compensated despite increasedlevels of IL-7. In addition, since abnormal MMD sequester all thegamma-c chains in a non functional state, this blocks the function ofthe other cytokines in this family.

The present invention further discloses the identification of the keyendogenous factor responsible for this abnormal state of the immunesystem in infected subjects and, more generally, responsible for adrastic modulation of the immune response in various pathophysiologicalconditions. Plasma samples from VP were indeed shown to contain anactivity—termed RIF—which is able to induce aberrant activation ofHealthy Donors (HD) CD4 T lymphocytes. RIF was found in all the plasmasamples of the VP examined. The pathophysiological significance of thisactivity was demonstrated by its absence in HIV Controller (HIC)patients where the IL-7/IL-7R system is normal and immune activation isbeneficial. RIF is also absent in the plasma of ART patients who havediminished their immune activation, restored IL-7R function andrecovered CD4 counts >500/mm³ (5).

RIF thus represents a major factor that controls the immune response,particularly through a modulation of CD4 T lymphocytes. It is remarkablethat RIF induces an aberrant pattern of activation in HD CD4 T cellsthat is undistinguishable from that observed directly ex vivo inpurified VP CD4 T cells. The invention further shows that RIF is thesecreted phospholipase A2 from Group I B (“PLA2 GIB”). The resultsdisclosed in this application show that (i) over expression of PLA2 GIBleads to a potent immunosuppression and that (ii) inhibition of PLA2 GIBleads to a remarkable increase or stimulation of immune function.GIBsPLA2 inhibitors were able to correct the inappropriate state of theimmune cells in plasma from subjects and can thus be used to treat(e.g., prevent, correct) immunodeficiency or immune disorders inmammals. GIBsPLA2 inhibition can also induce, stimulate, or helpmaintaining CD4 T cell counts and function, and thereby help stimulateefficient immune responses in patients. In particular, in HIV-infectedpatients, ART might be spared, or could be suspended, were anequilibrium to be reached between patient immune defenses and the virus.Were ART, given very early after infection as suggested by recentstudies, to be combined with RIF inhibitors, this would prevent anyRIF-induced alteration of the immune system. In addition, in the contextof some current failures of ART, patients with low CD4 counts afterprolonged ART may benefit from these inhibitors. Accordingly, theinvention provides methods for treating a subject by modulating GIBsPLA2expression or activity in the subject. More particularly, the inventionprovides a method for modulating an immune response in a subject in needthereof, comprising modulating GIBsPLA2 activity or expression in saidsubject.

The data provided in the examples also demonstrate that the presence ofRIF in the plasma of a subject indicates the HIV-induced pathogenesisstate of CD4 T cells. Accordingly, this invention provides methods ofmonitoring and/or diagnosing HIV infection in a subject by detecting thelevel of RIF in the plasma of the subject, among other things.

The data provided in the examples further demonstrate that the numberand/or size of membrane microdomains (MMD) on the T-cells of a subjectindicates the HIV-induced pathogenesis state of CD4 T cells.Accordingly, this disclosure also provides methods of monitoring and/ordiagnosing HIV infection in a subject by measuring the number and/orsize of membrane microdomains (MMD) on the T-cells of the subject, amongother things.

The data provided in the examples also indicate a role for RIF increating and/or maintaining the CD4 T cell disease state in HIV infectedsubjects. Accordingly, this disclosure also provides methods foridentifying a candidate HIV therapeutic agent that include measuringRIF-induced CD4 T cell activation in the presence of an agent. In someembodiments the methods comprise comparing the level of RIF-induced CD4T cell activation in the presence of the agent with the level ofRIF-induced CD4 T cell activation in the absence of the agent.

Definitions

The term “sequence identity” as applied to nucleic acid or proteinsequences, refers to the quantification (usually percentage) ofnucleotide or amino acid residue matches between at least two sequencesaligned using a standardized algorithm such as Smith-Waterman alignment(Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompsonet al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul etal. (1997) Nucleic Acids Res 25:3389-3402). BLAST2 may be used in astandardized and reproducible way to insert gaps in one of the sequencesin order to optimize alignment and to achieve a more meaningfulcomparison between them.

As used herein, “treatment” or “treat” refers to clinical interventionin an attempt to alter the natural course of the individual beingtreated, and can be performed either for preventive or curative purpose.Desirable effects of treatment include, but are not limited to,preventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, compositions andmethods of the invention are used to delay development of a disease ordisorder or to slow the progression of a disease or disorder.

The term “isolated”, as used herein, refers to molecules (e.g., nucleicor amino acid) that are removed from a component of their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated. An “isolated” polypeptide (orprotein) is for instance a polypeptide separated from a component of itsnatural environment and, preferably purified to greater than 90% or 95%purity as determined by, for example, electrophoretic (e.g., SDS-PAGE,isoelectric focusing (IEF), capillary electrophoresis) orchromatographic (e.g., ion exchange or reverse phase HPLC) migration. An“isolated” nucleic acid refers to a nucleic acid molecule separated froma component of its natural environment and/or assembled in a differentconstruct (e.g., a vector, expression cassette, recombinant host, etc.).

“Nucleic acid encoding an anti-GIBsPLA2 antibody” refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

A “subject” refers to a mammal. Examples of mammals include humans andnon-human animals such as, without limitation, domesticated animals(e.g., cows, sheep, cats, dogs, and horses), non-human primates (such asmonkeys), rabbits, and rodents (e.g., mice and rats).

The “modulation of an immune response” designates, within the context ofthe invention, any modification of the amount or activity or ratio ofimmune cells, preferably white blood cells (e.g., T lymphocytes, Blymphocytes, NK, NKT cells, macrophages, dendritic cells). In aparticular embodiment, modulating an immune response includes modulatingthe amount or activity of T lymphocytes, preferably of CD4-Tlymphocytes.

Refractory State Inducing Factor (RIF) or Phospholipase A2 Group IB

The term RIF is used interchangeably with Phospholipase A2 group IB,GIBsPLA2 (or PLA2 GIB). Phospholipase A2 group IB is a secreted proteinhaving a MW of from about 15 kDa and an isoelectric point of from about6.5 to about 8.0.

Within the context of the present invention, the term “GIBsPLA2” or“phospholipase A2 group TB” designates any native GIBsPLA2 protein fromany vertebrate source, including mammals such as primates (e.g. humans)and rodents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed GIBsPLA2, as well as any form ofGIBsPLA2 that results from processing inside or outside a cell. The termalso encompasses naturally-occurring variants of GIBsPLA2, e.g., splicevariants or allelic variants.

The amino acid sequence of an exemplary human GIBsPLA2 is shown below(SEQ ID NO: 2).

MKLLVLAVLL TVAAA DSGIS PRAVWQFRKM IKCVIPGSDPFLEYNNYGCY CGLGGSGTPV DELDKCCQTH DNCYDQAKKLDSCKFLLDNP YTHTYSYSCS GSAITCSSKN KECEAFICNCDRNAAICFSK APYNKAHKNL DTKKYCQS

Amino acids 1 to 15 of SEQ ID NO: 2 (underlined) are a signal sequence,and amino acids 16 to 22 of SEQ ID NO: 2 (in bold) are a propeptidesequence. The mature protein corresponds to amino acid residues 23-148of SEQ ID NO: 2, which is an exemplary processed human GIBsPLA2 protein.

Naturally-occurring variants include any protein comprising the sequenceof SEQ ID NO: 2, or the sequence of amino acid residues 23-148 of SEQ IDNO: 2, with one or more amino acid substitution, addition and/ordeletion of one or several (typically 1, 2 or 3) amino acid residues,preferably not more than 10 distinct amino acid substitution(s),addition(s), and/or deletion(s) of one or several (typically 1, 2 or 3)amino acid residues. Typical naturally-occurring variants retain abiological activity of SEQ ID NO: 2.

In this regard, in some embodiments, GIBsPLA2 has at least one activityselected from induction of formation of membrane microdomains (MMD) inCD4 T cells from healthy subjects, or rendering CD4 T cells of healthysubjects refractory to interleukin signaling, such as refractory to IL-2signaling or refractory to IL-7 signaling.

In some embodiments inducing formation of MMD comprises increasing thenumber of MMD on CD4 T cells of healthy subjects to at least about 80per cell, at least about 90 per cell, at least about 100 per cell, atleast about 110 per cell, or at least about 120 per cell. In anon-limiting preferred embodiment, inducing formation of MMD comprisesincreasing the number of MMD on CD4 T cells of healthy subjects to morethan 100 MMD per cell.

In some embodiments inducing formation of MMD comprises stimulatingformation of larger MMD than would otherwise be present on the CD4 Tcells. In some embodiments inducing formation of larger MMD comprisesstimulating formation MMD having a diameter of at least 100 nm, at least110 nm, at least 120 nm, at least 130 nm, or at least 140 nm. In anon-limiting preferred embodiment, inducing formation of larger MMDcomprises stimulating formation of MMD having a diameter larger than 120nm.

In some embodiments rendering CD4 T cells of healthy subjects refractoryto interleukin-7 signaling comprises a reduction of STATSA and/or Bphosphorylation in said cells by at least about 10%, at least about 20%,at least about 30%, or at least about 40%. In some embodiments renderingCD4 T cells of healthy subjects refractory to interleukin-7 signalingcomprises reducing the rate of nuclear translocation of phospho-STATSAand/or phospho-STATSB by at least about 20%, at least about 30%, atleast about 40%, or at least about 50%.

GIBsPLA2 activity may be measured by any suitable method known in theart, as illustrated in the examples, or later developed. GIBsPLA2activity may be measured in a plasma sample such as for example afractionated plasma sample, using e.g., ligand recruitment assays,immunoassays and/or enzymatic assays.

In a particular embodiment, the term GIBsPLA2 designates a humanprotein, particularly a protein comprising or having SEQ ID NO: 2, or anaturally-occurring variant thereof.

GIBsPLA2 according to this disclosure may be isolated, purified, and/orrecombinant. In certain embodiments, the invention may use, instead orin addition to a GIBsPLA2 protein, a nucleic acid encoding GIBsPLA2. Thenucleic acid may be DNA or RNA, single- or double-stranded.

An exemplary nucleic acid sequence encoding a GIBsPLA2 is shown in SEQID NO: 1 below.

ATGAAACTCCTTGTGCTAGCTGTGCTGCTCACAGTGGCCGCCGCCGACAGCGGCATCAGCCCTCGGGCCGTGTGGCAGTTCCGCAAAATGATCAAGTGCGTGATCCCGGGGAGTGACCCCTTCTTGGAATACAACAACTACGGCTGCTACTGTGGCTTGGGGGGCTCAGGCACCCCCGTGGATGAACTGGACAAGTGCTGCCAGACACATGACAACTGCTACGACCAGGCCAAGAAGCTGGACAGCTGTAAATTTCTGCTGGACAACCCGTACACCCACACCTATTCATACTCGTGCTCTGGCTCGGCAATCACCTGTAGCAGCAAAAACAAAGAGTGTGAGGCCTTCATTTGCAACTGCGACCGCAACGCTGCCATCTGCTTTTCAAAAGCTCCATATAACAAGGCACACAAGAACCTGGACACCAAGAAG TATTGTCAGAGTTGA

Alternative nucleic acid molecules encoding a GIBsPLA2 include anyvariant of SEQ ID NO:1 resulting from the degeneracy of the geneticcode, as well as any sequence which hybridizes to SEQ ID NO: 1 understringent conditions, more preferably having at least 80%, 85%, 90%, 95%or more sequence identity to SEQ ID NO; 1, and encoding a GIBsPLA2protein.

Method of Production of GIBsPLA2

GIBsPLA2 can be produced by any conventionally known protein expressionmethod and purification method. For example: (i) a method forsynthesizing peptides; (ii) a method for purifying and isolating themfrom the living body or cultured cells; or (iii) a method for producingthem with the use of genetic recombination techniques; and combinationsthereof and the like (for example, the standard techniques described forexample in Molecular Cloning (Sambrook, J., Fritsch, E. F., Maniatis,T., Cold Spring Harbor Laboratory Press) (1989) and Current Protocols inMolecular Biology (Ausubel, F. M., John Wiley and Sons, Inc. (1989)).

In a particular embodiment, the invention relates to a method forproducing GIBsPLA2 by expression of a coding nucleic acid in a hostcell, and collection or purification of GIBsPLA2. In this regard, theinvention also described recombinant host cells comprising a nucleicacid encoding a GIBsPLA2. Such cells may be prokaryotic (such asbacteria) or eukaryotic (such as yeast cells, insect cells, plant cellsor mammalian cells). The nucleic acid may be placed under the control ofany suitable regulatory sequence, such as a promoter, terminator, andthe like. Alternatively, the nucleic acid may be inserted in the hostcell in a location where expression is driven by an endogenous promoter.Techniques for inserting nucleic acids in cells are well known in theart.

GIBsPLA2 Modulation

The invention provides novel methods which comprise a modulation ofGIBsPLA2 in a subject in need thereof. The term “modulation” designatesany modification of the level (e.g., expression) or activity of GIBsPLA2in a subject. Also, modulation designates either an increase or adecrease GIBsPLA2 level or activity. A modulation more preferablydesignates a change by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%or more as compared to non-modulated situation. As a result, inhibitingGIBsPLA2 designates reducing by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80% or more GIBsPLA2 level or activity, as well as completelyblocking or suppressing GIBsPLA2 level or activity. Conversely,stimulating GIBsPLA2 designates increasing by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80% or more GIBsPLA2 level or activity. Depending onthe situation, the modulation may be transient, sustained or permanent.Also modulating the activity includes modulating the amount of GIBsPLA2in the subject, particularly in body fluids, modulating the potency ofthe protein (for instance by modulating the level of co-factors orsubstrate in the subject), and modulating the level or activity ofdegradation products produced by GIBsPLA2.

GIBsPLA2 Inhibition

In a particular embodiment, the invention provides compositions andmethods for inhibiting GIBsPLA2 in a subject. GIBsPLA2 inhibition may beobtained by the use of GIBsPLA2 inhibitors, i.e., any compound thatinhibit the expression or activity of GIBsPLA2. GIBsPLA2 inhibitorsinclude expression inhibitors, antagonists, sequestrators, or targetmasking compounds. Preferred types of GIBsPLA2 inhibitors includeGIBsPLA2 ligands (covalent or non-covalent), anti-GIBsPLA2 antibodies(and fragments and derivatives thereof), nucleic acids encodinganti-GIBsPLA2 antibodies (or fragments and derivatives thereof),inhibitory nucleic acids, peptides, or small drugs, or combination(s)thereof. Alternatively, or in addition, GIBsPLA2 inhibition can beobtained by vaccinating a subject against a GIBsPLA2 antigen, so thatantibodies are produced by the subject in need of PLA2-GIB inhibition.

Antibodies Against GIBsPLA2

Specific examples of GIBsPLA2 inhibitors are antibodies thatspecifically bind to GIBsPLA2.

Antibodies can be synthetic, monoclonal, or polyclonal and can be madeby techniques well known in the art. Such antibodies specifically bindvia the antigen-binding sites of the antibody (as opposed tonon-specific binding). GIBsPLA2 polypeptides, fragments, variants,fusion proteins, etc., can be employed as immunogens in producingantibodies immunoreactive therewith. More specifically, thepolypeptides, fragments, variants, fusion proteins, etc. containantigenic determinants or epitopes that elicit the formation ofantibodies.

These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hindrances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes can be identified by any of the methods known inthe art. Both polyclonal and monoclonal antibodies can be prepared byconventional techniques.

Preferred antibodies of the invention are directed to a GIBsPLA2epitope, and/or have been generated by immunization with a polypeptidecomprising a GIBsPLA2 epitope selected from: the mature GIBsPLA2protein, a fragment of GIBsPLA2 comprising at least 8 consecutive aminoacid residues of SEQ ID NO: 2 (or the corresponding residues of anatural variant of SEQ ID NO: 2), said fragment comprising at leastamino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid54, amino acid 71, or a combination thereof. Preferred antibodies of theinvention bind an epitope comprised between amino acid residues 50-71 ofSEQ ID NO: 2 or the corresponding residues of a natural variant of SEQID NO: 2.

The term “antibodies” is meant to include polyclonal antibodies,monoclonal antibodies, fragments thereof, such as F(ab′)2 and Fabfragments, single-chain variable fragments (scFvs), single-domainantibody fragments (VHHs or Nanobodies), bivalent antibody fragments(diabodies), as well as any recombinantly and synthetically producedbinding partners, human antibodies or humanized antibodies.

Antibodies are defined to be specifically binding preferably if theybind to GIBsPLA2 with a Ka of greater than or equal to about 10⁷ M-1.Affinities of antibodies can be readily determined using conventionaltechniques, for example those described by Scatchard et al., Ann. N.Y.Acad. Sci., 51:660 (1949).

Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, donkeys, goats, sheep, dogs,chickens, rabbits, mice, or rats, using procedures that are well knownin the art. In general, purified GIBsPLA2 or a peptide based on theamino acid sequence of GIBsPLA2 that is appropriately conjugated isadministered to the host animal typically through parenteral injection.The immunogenicity of GIBsPLA2 can be enhanced through the use of anadjuvant, for example, Freund's complete or incomplete adjuvant.Following booster immunizations, small samples of serum are collectedand tested for reactivity to GIBsPLA2 polypeptide. Examples of variousassays useful for such determination include those described inAntibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; as well as procedures, such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA),dot blot assays, and sandwich assays. See U.S. Pat. Nos. 4,376,110 and4,486,530.

Monoclonal antibodies can be readily prepared using well knownprocedures. See, for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKeam, and Bechtol (eds.), 1980.

For example, the host animals, such as mice, can be injectedintraperitoneally at least once and preferably at least twice at about 3week intervals with isolated and purified wild-type or mutant GIBsPLA2protein or conjugated GIBsPLA2 peptide, optionally in the presence ofadjuvant. Mouse sera are then assayed by conventional dot blot techniqueor antibody capture (ABC) to determine which animal is best to fuse.Approximately two to three weeks later, the mice are given anintravenous boost of protein or peptide. Mice are later sacrificed andspleen cells fused with commercially available myeloma cells, such asAg8.653 (ATCC), following established protocols. Briefly, the myelomacells are washed several times in media and fused to mouse spleen cellsat a ratio of about three spleen cells to one myeloma cell. The fusingagent can be any suitable agent used in the art, for example,polyethylene glycol (PEG). Fusion is plated out into plates containingmedia that allows for the selective growth of the fused cells. The fusedcells can then be allowed to grow for approximately eight days.Supernatants from resultant hybridomas are collected and added to aplate that is first coated with goat anti-mouse Ig. Following washes, alabel, such as a labeled GIBsPLA2 polypeptide, is added to each wellfollowed by incubation. Positive wells can be subsequently detected.Positive clones can be grown in bulk culture and supernatants aresubsequently purified over a Protein A column (Pharmacia).

The monoclonal antibodies of the disclosure can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3:1-9 (1990), which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7:394 (1989).

Antigen-binding fragments of such antibodies, which can be produced byconventional techniques, are also encompassed by the present invention.Examples of such fragments include, but are not limited to, Fab andF(ab′)2 fragments. Antibody fragments and derivatives produced bygenetic engineering techniques are also provided.

The monoclonal antibodies of the present disclosure include chimericantibodies, e.g., humanized versions of murine monoclonal antibodies.Such humanized antibodies can be prepared by known techniques, and offerthe advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment, a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment can comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806.

Antibodies produced by genetic engineering methods, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,can be used. Such chimeric and humanized monoclonal antibodies can beproduced by genetic engineering using standard DNA techniques known inthe art, for example using methods described in Robinson et al.International Publication No. WO 87/02671; Akira, et al. European PatentApplication 0184187; Taniguchi, M., European Patent Application 0171496;Morrison et al. European Patent Application 0173494; Neuberger et al.PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat.No. 4,816,567; Cabilly et al. European Patent Application 0125023;Better et al., Science 240:1041 1043, 1988; Liu et al., PNAS 84:34393443, 1987; Liu et al., J. Immunol. 139:3521 3526, 1987; Sun et al. PNAS84:214 218, 1987; Nishimura et al., Canc. Res. 47:999 1005, 1987; Woodet al., Nature 314:446 449, 1985; and Shaw et al., J. Natl. Cancer Inst.80:1553 1559, 1988); Morrison, S. L., Science 229:1202 1207, 1985; Oi etal., BioTechniques 4:214, 1986; Winter U.S. Pat. No. 5,225,539; Jones etal., Nature 321:552 525, 1986; Verhoeyan et al., Science 239:1534, 1988;and Beidler et al., J. Immunol. 141:4053 4060, 1988.

In connection with synthetic and semi-synthetic antibodies, such termsare intended to cover but are not limited to antibody fragments, isotypeswitched antibodies, humanized antibodies (e.g., mouse-human,human-mouse), hybrids, antibodies having plural specificities, and fullysynthetic antibody-like molecules.

For therapeutic applications, “human” monoclonal antibodies having humanconstant and variable regions are often preferred so as to minimize theimmune response of a patient against the antibody. Such antibodies canbe generated by immunizing transgenic animals which contain humanimmunoglobulin genes. See Jakobovits et al. Ann NY Acad Sci 764:525-535(1995).

Human monoclonal antibodies against GIBsPLA2 polypeptides can also beprepared by constructing a combinatorial immunoglobulin library, such asa Fab phage display library or a scFv phage display library, usingimmunoglobulin light chain and heavy chain cDNAs prepared from mRNAderived from lymphocytes of a subject. See, e.g., McCafferty et al. PCTpublication WO 92/01047; Marks et al. (1991) J. Mol. Biol. 222:581 597;and Griffiths et al. (1993) EMBO J 12:725 734. In addition, acombinatorial library of antibody variable regions can be generated bymutating a known human antibody. For example, a variable region of ahuman antibody known to bind GIBsPLA2, can be mutated by, for example,using randomly altered mutagenized oligonucleotides, to generate alibrary of mutated variable regions which can then be screened to bindto GIBsPLA2. Methods of inducing random mutagenesis within the CDRregions of immunoglobin heavy and/or light chains, methods of crossingrandomized heavy and light chains to form pairings and screening methodscan be found in, for example, Barbas et al. PCT publication WO 96/07754;Barbas et al. (1992) Proc. Nat'l Acad. Sci. USA 89:4457 4461.

An immunoglobulin library can be expressed by a population of displaypackages, preferably derived from filamentous phage, to form an antibodydisplay library. Examples of methods and reagents particularly amenablefor use in generating antibody display library can be found in, forexample, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTpublication WO 92/18619; Dower et al. PCT publication WO 91/17271;Winter et al. PCT publication WO 92/20791; Markland et al. PCTpublication WO 92/15679; Breitling et al. PCT publication WO 93/01288;McCafferty et al. PCT publication WO 92/01047; Garrard et al. PCTpublication WO 92/09690; Ladner et al. PCT publication WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370 1372; Hay et al. (1992) HumAntibody Hybridomas 3:81 85; Huse et al. (1989) Science 246:1275 1281;Griffiths et al. (1993) supra; Hawkins et al. (1992) J Mol Biol 226:889896; Clackson et al. (1991) Nature 352:624 628; Gram et al. (1992) PNAS89:3576 3580; Garrad et al. (1991) Bio/Technology 9:1373 1377;Hoogenboom et al. (1991) Nuc Acid Res 19:4133 4137; and Barbas et al.(1991) PNAS 88:7978 7982. Once displayed on the surface of a displaypackage (e.g., filamentous phage), the antibody library is screened toidentify and isolate packages that express an antibody that binds aGIBsPLA2 polypeptide. In a preferred embodiment, the primary screeningof the library involves panning with an immobilized GIBsPLA2 polypeptideand display packages expressing antibodies that bind immobilizedGIBsPLA2 polypeptide are selected.

In a particular embodiment, the invention relates to a compositioncomprising an anti-GIBsPLA2 antibody (or a fragment or derivativethereof) and a pharmaceutically acceptable excipient.

Existing anti-Phospholipase A2-GIB monoclonal antibodies include MabCH-7 (Labome), MAB35018 (Labome), EPR5186 (Genetex); LS-C138332(Lifespan) or CABT-17153MH (creative biomart). Examples of polyclonalantibodies include for instance N1C3 from GeneTex. As indicated above,preferred anti-GIBsPLA2 antibodies of the invention bind matureGIBsPLA2, even more preferably an epitope comprised in a domain ofGIBsPLA2 comprising an amino acid selected from amino acid 70, aminoacid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, ora combination thereof. Preferred antibodies of the invention bind anepitope comprised between amino acid residues 50-71 of SEQ ID NO: 2 orthe corresponding residues of a natural variant of SEQ ID NO: 2.

In an alternative embodiment, the invention relates to and uses acomposition comprising a nucleic acid encoding an anti-GIBsPLA2 antibody(or a fragment or derivative thereof) and a pharmaceutically acceptableexcipient.

Inhibitory Nucleic Acids

In an alternative embodiment, the GIBsPLA2 inhibitor is an inhibitorynucleic acid, i.e., any nucleic acid molecule which inhibits GIBsPLA2gene or protein expression. Preferred inhibitory nucleic acids includeantisense nucleic acids, short interfering RNAs (siRNAs), small hairpinRNAs (shRNA), microRNAs, aptamers, or ribozymes. In a particularembodiment, the inhibitory nucleic acid is a small interfering RNA thatprevents translation of GIBsPLA2 mRNA. In another particular embodiment,the inhibitory nucleic acid is an antisense oligonucleotide thatprevents translation of GIBsPLA2 mRNA. In another particular embodiment,the inhibitory nucleic acid is a small hairpin RNA that preventstranslation of GIBsPLA2 mRNA.

siRNA comprise a sense nucleic acid sequence and an anti-sense nucleicacid sequence of the polynucleotide of interest. siRNA are constructedsuch that a single transcript (double stranded RNA) have both the senseand complementary antisense sequences from the target gene. Thenucleotide sequence of siRNAs may be designed using an siRNA designcomputer program available from, for example, the Ambion website on theworld wide web.

In some embodiments, the length of the antisense oligonucleotide orsiRNAs is less than or equal to 10 nucleotides. In some embodiments, thelength of the antisense oligonucleotides and siRNAs is as long as thenaturally occurring transcript. In some embodiments, the antisenseoligonucleotides and siRNAs have 18-30 nucleotides. In some embodiments,the antisense oligonucleotides and siRNAs are less than 25 nucleotidesin length.

Preferred inhibitory nucleic acid molecules comprise a domain having anucleotide sequence that is perfectly complementary to a region of aGIBsPLA2 gene or RNA. Such a domain contains typically from 4 to 20nucleotides, allowing specific hybridization and optimal inhibition thegene transcription or RNA translation. The sequence of the inhibitorynucleic acids may be derived directly from the sequence of a geneencoding GIBsPLA2, such as SEQ ID NO: 1. Alternatively, or in addition,inhibitory nucleic acids may hybridize to a regulatory element in aGIBsPLA2 gene or RNA, such as a promoter, a splicing site, etc., andprevent effective regulation thereof.

Specific examples of inhibitory nucleic acid molecules of the presentinvention include isolated single strand nucleic acid moleculesconsisting of from 10 to 50 consecutive nucleotides of SEQ ID NO: 1.Specific examples of inhibitory nucleic acid molecules of the inventionare antisense nucleic acids consisting of the following nucleotidesequence or the perfectly complementary strand thereof:

(SEQ ID NO: 3) ATGAAACTCCTTGTGCTAG (SEQ ID NO: 4) ACAGCGGCATCAGC(SEQ ID NO: 5) TTCCGCAAAATGATCAA (SEQ ID NO: 6) CCCGGGGAGTGACCCC(SEQ ID NO: 7) TACGGCTGCTACTGTGGCTT (SEQ ID NO: 8)GACACATGACAACTGCTACGACC (SEQ ID NO: 9) ACCCACACCTATTCATACTCGT(SEQ ID NO: 10) ATCACCTGTAGCAGCA (SEQ ID NO: 11) AGCTCCATATAACAAGGCA(SEQ ID NO: 12) CAAGAAGTATTGTCAGAG

Peptide and Small Drugs

In an alternative embodiment, the GIBsPLA2 inhibitor is a peptide orsmall drug that inhibits the activity of GIBsPLA2. The peptide or smalldrug is typically a molecule that selectively binds GIBsPLA2, or asubstrate of GIBsPLA2, or a co-factor of GIBsPLA2, or a degradationproduct or metabolite of GIBsPLA2 pathway.

Peptides preferably contain from 3 to 20 amino acid residues, and theirsequence may be identical to a domain of GIBsPLA2 (bait peptide) or to adomain of a GIBsPLA2 substrate, co-factor, degradation product ormetabolite. Preferred peptides of the invention contain from 4 to 30consecutive amino acid residues of SEQ ID O: 2 (or of a correspondingsequence of a natural variant of SEQ ID NO: 2). Most preferred peptidesof the invention comprise from 5 to 25 consecutive amino acid residuesof SEQ ID O: 2 (or of a corresponding sequence of a natural variant ofSEQ ID NO: 2) and further comprise at least one of the following aminoacid residues of SEQ ID O: 2 (or of a corresponding sequence of anatural variant of SEQ ID NO: 2): amino acid 70, amino acid 121, aminoacid 50, amino acid 52, amino acid 54, amino acid 71, or a combinationthereof. Specific examples of peptides of the invention are peptides ofless than 25 amino acids comprising anyone of the following sequences:

(SEQ ID NO: 13) NNYGCY (SEQ ID NO: 14) CYCGLG (SEQ ID NO: 15)YNNYGCYCGLGGSG (SEQ ID NO: 16) FLEYNNYGCYCGLGGSGTPV (SEQ ID NO: 17)QTHDN (SEQ ID NO: 18) CQTHDNC (SEQ ID NO: 19) ECEAFICNC (SEQ ID NO: 20)DRNAAI (SEQ ID NO: 21) DRNAAICFSKAPYNKAHKNL

The peptides of the invention can comprise peptide, non-peptide and/ormodified peptide bonds. In a particular embodiment, the peptidescomprise at least one peptidomimetic bond selected from intercalation ofa methylene (—CH₂—) or phosphate (—PO₂—) group, secondary amine (—NH—)or oxygen (—O—), alpha-azapeptides, alpha-alkylpeptides,N-alkylpeptides, phosphonamidates, depsipeptides, hydroxymethylenes,hydroxyethylenes, dihydroxyethylenes, hydroxyethylamines, retro-inversopeptides, methyleneoxy, cetomethylene, esters, phosphinates,phosphinics, or phosphonamides. Also, the peptides may comprise aprotected N-ter and/or C-ter function, for example, by acylation, and/oramidation and/or esterification.

The peptides of the invention may be produced by techniques known per sein the art such as chemical, biological, and/or genetic synthesis.

Each of these peptides, in isolated form, represents a particular objectof the present invention.

Preferred small drugs are hydrocarbon compounds that selectively bindGIBsPLA2.

Small drugs and peptides are preferably obtainable by a methodcomprising: (i) contacting a test compound with GIBsPLA2 or a fragmentthereof, (ii) selecting a test compound which binds GIBsPLA2 or saidfragment thereof, and (iii) selecting a compound of (ii) which inhibitsan activity of GIBsPLA2. Such a method represents a particular object ofthe invention.

Small drugs and peptides are also obtainable by a method comprising: (i)contacting a test compound with a GIBsPLA2 substrate, co-factor, ordegradation product, or a fragment thereof, (ii) selecting a testcompound which binds to said GIBsPLA2 substrate, co-factor, ordegradation product, or a fragment thereof, and (iii) selecting acompound of (ii) which inhibits an activity of GIBsPLA2. Such a methodrepresents a particular object of the invention.

GIBsPLA2 Soluble Receptors

In an alternative embodiment, the GIBsPLA2 inhibitor is a soluble formof a GIBsPLA2 receptor. Such soluble receptor compounds are able to bindGIBsPLA2, thereby inhibiting its activity by acting as a bait or maskingagent.

A specific embodiment of such inhibitors is a soluble form of a human ormurine GIBsPLA2 receptor, or a GIBsPLA2-binding fragment thereof.

The amino acid sequences of murine and human soluble receptors aredepicted in SEQ ID NOs: 22 and 23, respectively. The term solublereceptor thus encompasses any GIBsPLA2-binding polypeptide comprisingall or a fragment of the sequence of SEQ ID NO: 22 or 23.

A GIBsPLA2-binding fragment designates any fragment of such apolypeptide comprising preferably at least 5 consecutive amino acidresidues thereof, more preferably at least 8, 10, or 12, which bindsPLA2GIB specifically. Specific binding of the receptor moleculeindicates that the receptor molecule binds to PLA2GIB with higheraffinity (e.g., by at least 5 fold) than to PLA2-IIA or IID. A fragmentas defined above most preferably comprises less than 50 amino acidresidues.

Examples of GIBsPLA2-binding polypeptides are, without limitation,polypeptides comprising at least one of the following amino acidsequences:

(SEQ ID NO: 24) LSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKW(SEQ ID NO: 25) WEKDLNSHICYQFNLLS (SEQ ID NO: 26)DCESTLPYICKKYLNHIDHEIVEK (SEQ ID NO: 27)QYKVQVKSDNTVVARKQIHRWIAYTSSGGDICE (SEQ ID NO: 28)LSYLNWSQEITPGPFVEHHCGTLEVVSA (SEQ ID NO: 29) SRFEQAFITSLISSVAEKDSYFW(SEQ ID NO: 30) WICRIPRDVRPKFPDWYQYDAPWLFYQNA (SEQ ID NO: 31)AFHQAFLTVLLSRLGHTHWIGLSTTDNGQT

SEQ ID NOs: 24-26 derive from the sequence of human soluble PLA2GIBreceptor, while SEQ ID NOs: 27-31 derive from the sequence of murinesoluble PLA2GIB receptor.

Vaccination

In an alternative (or cumulative) embodiment, inhibition of GIBsPLA2 ina subject is obtained by vaccinating (or immunizing) the subject with aGIBsPLA2 antigen. As a result of such a vaccination or immunization, thesubject produces antibodies (or cells) which inhibit GIBsPLA2. Inparticular, injection(s) of a GIBsPLA2 antigen (e.g., an immunogenicGIBsPLA2 essentially devoid of biological activity) can generateantibodies in the treated subject. These antibodies will protect againstan excess of GIBsPLA2 expression and can be used along as immunotherapyor a vaccine prophyllaxy.

An object of the invention thus resides in a method of vaccinating asubject comprising administering to the subject a GIBsPLA2 antigen.

A further object of the invention relates to a GIBsPLA2 antigen for useto vaccinate a subject in need thereof.

In a particular embodiment, the GIBsPLA2 antigen used for vaccination isan inactivated immunogenic molecule that induces an immune responseagainst GIBsPLA2 in a subject. Inactivation may be obtained e.g., bychemically or physically altering GIBsPLA2 or by mutating or truncatingthe protein, or both; and immunogenicity may be obtained as a result ofthe inactivation and/or by further conjugating the protein to a suitablecarrier or hapten, such as KLH, HSA, polylysine, a viral anatoxin, orthe like, and/or by polymerization, or the like. The antigen may thus bechemically or physically modified, e.g., to improve its immunogenicity.

In a preferred embodiment, the GIBsPLA2 antigen of the inventioncomprises GIBsPLA2 or an epitope-containing fragment or mimotopethereof.

In a particular embodiment, the GIBsPLA2 antigen comprises a full lengthGIBsPLA2 protein. In a further particular embodiment, the GIBsPLA2antigen comprises a protein comprising SEQ ID NO: 2, or a sequencehaving at least 90% identity to SEQ ID NO: 2.

In an alternative embodiment, the GIBsPLA2 antigen comprises a fragmentof a GIBsPLA2 protein comprising at least 6 consecutive amino acidresidues and containing an immunogenic epitope, or a mimotope thereof.In a preferred embodiment, the GIBsPLA2 antigen comprises at least from6 to 20 amino acid residues. Preferred peptides of the invention containfrom 4 to 30 consecutive amino acid residues of SEQ ID O: 2 (or of acorresponding sequence of a natural variant of SEQ ID NO: 2). Mostpreferred peptides of the invention comprise from 5 to 25 consecutiveamino acid residues of SEQ ID O: 2 (or of a corresponding sequence of anatural variant of SEQ ID NO: 2) and further comprise at least one ofthe following amino acid residues of SEQ ID O: 2 (or of a correspondingsequence of a natural variant of SEQ ID NO: 2): amino acid 70, aminoacid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, ora combination thereof. Specific examples of peptides of the inventionare peptides of less than 50 amino acids comprising anyone of thefollowing sequences:

(SEQ ID NO: 13) NNYGCY (SEQ ID NO: 14) CYCGLG (SEQ ID NO: 15)YNNYGCYCGLGGSG (SEQ ID NO: 16) FLEYNNYGCYCGLGGSGTPV (SEQ ID NO: 17)QTHDN (SEQ ID NO: 18) CQTHDNC (SEQ ID NO: 19) ECEAFICNC (SEQ ID NO: 20)DRNAAI (SEQ ID NO: 21) DRNAAICFSKAPYNKAHKNL

The GIBsPLA2 antigen may be in various forms such as in free form,polymerized, chemically or physically modified, and/or coupled (i.e.,linked) to a carrier molecule. Coupling to a carrier may increase theimmunogenicity and (further) suppress the biological activity of theGIBsPLA2 polypeptide. In this regard, the carrier molecule may be anycarrier molecule or protein conventionally used in immunology such asfor instance KLH (Keyhole limpet hemocyanin), ovalbumin, bovine serumalbumin (BSA), a viral or bacterial anatoxin such as toxoid tetanos,toxoid diphteric B cholera toxin, mutants thereof such as diphtheriatoxin CRM 197, an outer membrane vesicle protein, a polylysine molecule,or a virus like particle (VLP). In a preferred embodiment, the carrieris KLH or CRM197 or a VLP.

Coupling of GIBsPLA2 to a carrier may be performed by covalent chemistryusing linking chemical groups or reactions, such as for instanceglutaraldehyde, biotin, etc. Preferably, the conjugate or the GIBsPLA2protein or fragment or mimotope is submitted to treatment withformaldehyde in order to complete inactivation of GIBsPLA2.

In a particular embodiment, the GIBsPLA2 antigen comprises a full lengthGIBsPLA2 protein, optionally coupled to a carrier protein. In apreferred embodiment, the GIBsPLA2 antigen comprises a proteincomprising SEQ ID NO: 2, or a sequence having at least 90% identity toSEQ ID NO: 2, coupled to a carrier protein.

In another particular embodiment, the GIBsPLA2 antigen comprises animmunogenic peptide or mimotope of GIBsPLA2, optionally coupled to acarrier protein. In a more preferred embodiment, the GIBsPLA2 antigencomprises a polypeptide of at least 10 amino acids long comprising atleast one of the following amino acid residues of SEQ ID O: 2 (or of acorresponding sequence of a natural variant of SEQ ID NO: 2): amino acid70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, aminoacid 71, or a combination thereof, optionally coupled to a carriermolecule.

The immunogenicity of the GIBsPLA2 antigen may be tested by variousmethods, such as by immunization of a non-human animal grafted withhuman immune cells, followed by verification of the presence ofantibodies, or by sandwich ELISA using human or humanized antibodies.The lack of biological activity may be verified by any of the activitytests described in the application. In a preferred embodiment, theGIBsPLA2 antigen has less than 20%, more preferably less than 15%, 10%,5% or even 1% of the activity of a wild-type GIBsPLA2 protein in an invitro method of (i) induction of formation of membrane microdomains(MMD) in CD4 T cells or (ii) in rendering CD4 T cells refractory to IL-2signaling or refractory to IL-7 signaling.

In a particular embodiment, the invention relates to an inactivated andimmunogenic GIBsPLA2.

In a further particular embodiment, the invention relates to a GIBsPLA2protein or a fragment or mimotope thereof conjugated to a carriermolecule, preferably to KLH.

In a further aspect, the invention relates to a vaccine comprising aGIBsPLA2 antigen, a suitable excipient and, optionally, a suitableadjuvant.

Such molecules and conjugates and vaccines represent potent agents foruse to immunize subjects, thereby causing a sustained GIBsPLA2inhibition. Upon repetition, such methods can be used to cause apermanent GIBsPLA2 inhibition.

A further object of the invention relates to of a method for inducingthe production of antibodies that neutralize the activity of endogenousGIBsPLA2 in a subject in need thereof, the method comprisingadministering to said subject a therapeutically effective amount of aGIBsPLA2 antigen or vaccine.

Administration of an antigen or vaccine of the invention may be by anysuitable route, such as by injection, preferably intramuscular,subcutaneous, transdermal, intravenous or intraarterial; by nasal, oral,mucosal or rectal administration.

The GIBsPLA2 antigen or vaccine may be used for treating any diseaselinked to an over-production of GIBsPLA2. More specifically, thisinvention relates to a method for treating a disease linked to anover-production of GIBsPLA2 in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of aGIBsPLA2 antigen or of a vaccine composition comprising a GIBsPLA2antigen.

GIBsPLA2 Agonists or Activators

The term GIBsPLA2 “agonist”, within the context of the presentinvention, encompasses any substance having, or mediating orup-regulating GIBsPLA2 activity such as, without limitation, a peptide,a polypeptide, a recombinant protein, a conjugate, a natural orartificial ligand, a degradation product, a homolog, a nucleic acid,DNA, RNA, an aptamer, etc., or a combination thereof. The term “agonist”encompasses both full and partial agonists. A particular example of aGIBsPLA2 agonist is a GIBsPLA2 protein or a nucleic acid encoding aGIBsPLA2 protein.

In a particular embodiment, the invention relates to methods forinhibiting an immune response in a subject, comprising administering tothe subject a GIBsPLA2 protein or a nucleic acid encoding a GIBsPLA2protein.

Compositions

The invention also relates to compositions comprising a GIBsPLA2modulator or antigen as herein described as an active ingredient, andpreferably a pharmaceutically acceptable carrier.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention (active ingredient) and a medium generally accepted in theart for the delivery of biologically active compounds to the subject inneed thereof. Such a carrier includes all pharmaceutically acceptablecarriers, diluents, medium or supports therefore. Conventionalpharmaceutical practice may be employed to provide suitable formulationsor compositions to subjects, for example in unit dosage form.

The compounds or compositions according to the invention may beformulated in the form of ointment, gel, paste, liquid solutions,suspensions, tablets, gelatin capsules, capsules, suppository, powders,nasal drops, or aerosol, preferably in the form of an injectablesolution or suspension. For injections, the compounds are generallypackaged in the form of liquid suspensions, which may be injected viasyringes or perfusions, for example. In this respect, the compounds aregenerally dissolved in saline, physiological, isotonic or bufferedsolutions, compatible with pharmaceutical use and known to the personskilled in the art. Thus, the compositions may contain one or moreagents or excipients selected from dispersants, solubilizers,stabilizers, preservatives, etc. Agents or excipients that can be usedin liquid and/or injectable formulations are notably methylcellulose,hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80,mannitol, gelatin, lactose, vegetable oils, acacia, etc. The carrier canalso be selected for example from methyl-beta-cyclodextrin, a polymer ofacrylic acid (such as carbopol), a mixture of polyethylene glycol andpolypropylene glycol, monoethanolamine and hydroxymethyl cellulose.

The compositions generally comprise an effective amount of a compound ofthe invention, e.g., an amount that is effective to modulate GIBsPLA2.Generally, the compositions according to the invention comprise fromabout 1 μg to 1000 mg of a GIBsPLA2 modulator, such as from 0.001-0.01,0.01-0.1, 0.05-100, 0.05-10, 0.05-5, 0.05-1, 0.1-100, 0.1-1.0, 0.1-5,1.0-10, 5-10, 10-20, 20-50, and 50-100 mg, for example between 0.05 and100 mg, preferably between 0.05 and 5 mg, for example 0.05, 0.1, 0.2,0.3, 0.4, 0.5, 1, 2, 3, 4 or 5 mg. The dosage may be adjusted by theskilled person depending on the modulator and the disease.

The compositions of the invention can further comprise one or moreadditional active compounds, for simultaneous or sequential use.

The invention also relates to a method for preparing a pharmaceuticalcomposition, comprising mixing a GIBsPLA2 modulator as previouslydescribed and a pharmaceutically acceptable excipient, and formulatingthe composition in any suitable form or container (syringe, ampoule,flask, bottle, pouch, etc.).

The invention also relates to a kit comprising (i) a compositioncomprising a GIBsPLA2 modulator as previously described, (ii) at leastone container, and optionally (iii) written instructions for using thekit.

Diseases

The compounds and compositions of the invention may be used to treat anydisease related to an inappropriate (e.g., defective or improper) immuneresponse, particularly to an inappropriate CD4 T cell activity, as wellas any disease where an increased immunity may ameliorate the subjectcondition. These diseases are sometime referred to as “immune disorders”in the present application. This includes immunodefective situations(e.g., caused by viral infection, pathogenic infection, cancer, etc.),autoimmune diseases, grafts, diabetes, inflammatory diseases, cancers,allergies, asthma, psoriasis, urticaria, eczema and the like.

Immunodeficiencies and Associated Disorders

In a first aspect, the invention is based on an inhibition of GIBsPLA2in a subject, thereby increasing or restoring an immune activity,particularly a CD4-T cell-mediated activity.

In a particular embodiment, the invention is therefore directed tomethods for stimulating an immune response in a subject in need thereof,comprising inhibiting GIBsPLA2 in said subject.

In a particular embodiment, the invention is directed to methods formodulating white blood cells in a subject in need thereof, comprisinginhibiting GIBsPLA2 in said subject.

Examples of diseases that can benefit from GIBsPLA2 inhibitors are alldiseases with an immunodeficiency such as HIV-mediated immunodeficiency.In this regard, in a particular embodiment, the invention is directed tomethods for treating an immunodeficiency or an associated disorder in asubject in need thereof, comprising inhibiting GIBsPLA2 in said subject.

In another particular embodiment, the invention is directed to aGIBsPLA2 inhibitor for use for treating an immunodeficiency or anassociated disorder in a subject in need thereof.

Immunodeficiencies and associated disorders designate any condition orpathology characterized by and/or caused by a reduced immune function orresponse in a subject. Immunodeficiencies may be caused by e.g., viralinfection (e.g., HIV, hepatitis B, etc.), bacterial infection, cancer,or other pathological conditions. The term “immunodeficiency-associateddisorder” therefore designates any disease caused by or associated withan immunodeficiency. The invention is particularly suitable for treatingimmunodeficiencies related to CD4-T cells, and associated diseases. Thepresent application indeed demonstrates that the biological effects ofGIBsPLA2 are involved in CD4 T cell disease state. Accordingly, blockingthe activity of GIBsPLA2 has a therapeutic benefit in subjects withaltered response to cytokine causing immunodeficiency as often observedin patients infected with HIV.

Accordingly, in a particular embodiment, the invention relates tomethods of treating HIV infection in a subject by inhibiting GIBsPLA2 inthe subject, preferably by administering a GIBsPLA2 inhibitor or vaccineto the subject. In some embodiments the subject is an early HIV patientand the methods results in increasing the probability that the patientis a HIV controller. In some embodiments the subject is a patient withlow immunoreconstitution after antiretroviral treatment and/or withsevere idiopathic CD4 T lymphopenia (ICL). The invention also relates toa method for increasing CD4-T cell activity in a HIV-infected subject byinhibiting GIBsPLA2 in the subject, preferably by administering aGIBsPLA2 inhibitor or vaccine to the subject.

In another embodiment, the invention relates to methods of treatingacute and/or chronic inflammation and processes derived frominflammatory reactions in a subject by injecting GIBsPLA2 in thesubject, either directly or associated with anti-inflammatory drugs.

The invention also provides methods for treating cancer by increasing animmune response in the subject, comprising inhibiting GIBsPLA2 in thesubject, preferably by administering a GIBsPLA2 inhibitor or vaccine tothe subject. The invention also provides methods of treating CD4 Tcell-linked immunodeficiency associated with cancer in a subject byinhibiting GIBsPLA2 in the subject, preferably by administering aGIBsPLA2 inhibitor or vaccine to the subject.

Pathologic Immune Responses and Associated Diseases

The invention may be used to treat any disease related to aninappropriate (e.g., pathologic or improper) immune response or to anundesirable (hyper)activity or (hyper)activation of the immune system,particularly to an inappropriate CD4 T cell activity. These diseasesinclude, for instance, autoimmune diseases, grafts, diabetes, allergies,asthma, psoriasis, urticaria, eczema and the like.

In a further aspect, the invention is thus based on an activation orinduction of GIBsPLA2 in a subject, thereby inhibiting an immuneactivity, particularly a CD4-T cell-mediated activity.

In a particular embodiment, the invention is therefore directed tomethods for inhibiting an immune response in a subject in need thereof,comprising inducing or activating GIBsPLA2 in said subject.

In a particular embodiment, the invention is directed to methods forinhibiting white blood cells in a subject in need thereof, comprisinginhibiting GIBsPLA2 in said subject.

In another particular embodiment, the invention is directed to methodsfor treating disorder caused by an undesirable immune response in asubject in need thereof, comprising inducing or activating GIBsPLA2 insaid subject.

Inducing or activating GIBsPLA2 in a subject preferably comprisesadministering to the subject a GIBsPLA2 agonist, for example a GIBsPLA2protein or a functional fragment thereof.

In another particular embodiment, the invention is directed to aGIBsPLA2 agonist or activator for use for treating a disorder caused byan undesirable immune response in a subject in need thereof.

Examples of diseases that can benefit from GIBsPLA2 agonists areautoimmune disorders, cancers, viral diseases, bacterial infections,etc.

In a particular embodiment, the invention is directed to methods fortreating an auto-immune disorder in a subject in need thereof,comprising stimulating or inducing GIBsPLA2 in said subject.

In another particular embodiment, the invention is directed to acompound or a composition of the invention for use in treating anauto-immune disorder in a subject in need thereof.

In a particular embodiment, the invention is directed to methods fortreating a cancer in a subject in need thereof, comprising stimulatingor inducing GIBsPLA2 in said subject.

In another particular embodiment, the invention is directed to acompound or a composition of the invention for use in treating cancer ina subject in need thereof.

Another particular embodiment of the invention relates to a method fortreating (e.g., reducing or preventing or inhibiting) graft rejection,or for treating graft vs host disease in a transplanted subject,comprising stimulating or inducing GIBsPLA2 in said subject. A furtherobject of the invention is a method for improving allogeneic grafttolerance in a subject comprising stimulating or inducing GIBsPLA2 insaid subject.

Anti-Microbial Activity

The present application also provides, in a further aspect, a method forkilling microbes using GIBsPLA2. By acting directly on the membranes,GIBsPLA2 can destroy or kill bacteria, enveloped viruses, parasites andthe like.

In acute infections or in infections, GIBsPLA2 may be used either aloneor associated with antibiotics, anti-viral, anti-retroviral andanti-parasite drugs. In the case of microbes resistant to knownanti-microbial drugs, GIBsPLA2 may represent an alternative therapy. Itcan be used in very short term treatment, e.g., in very dangerous andacute clinical situations.

Specific examples of diseases that can benefit from treatment byGIBsPLA2 according to the invention are all the clinical situations withan hyper activity of the immune system or a chronic inflammation such asMultiple sclerosis, Myasthenia gravis, Autoimmune neuropathies such asGuillain-Barré, Autoimmune uveitis, Uveitis, Autoimmune hemolyticanemia, Pernicious anemia, Autoimmune thrombocytopenia, Temporalarteritis, Anti-phospholipid syndrome, Vasculitides such as Wegener'sgranulomatosis, Behcet's disease, Atherosclerosis, Psoriasis, Dermatitisherpetiformis, Pemphigus vulgaris, Vitiligo, Pemphigus Vulgaris, MycosisFungoides, Allergic Contact Dermatitis, Atopic Dermatitis, LichenPlanus, PLEVA, eczema, Crohn's Disease, Ulcerative colitis, Primarybiliary cirrhosis, Autoimmune hepatitis, Type 1 diabetes mellitus,Addison's Disease, Grave's Disease, Hashimoto's thyroiditis, Autoimmuneoophoritis and orchitis, Autoimmune Thyroiditis, Rheumatoid arthritis,Systemic lupus erythematosus, Scleroderma, Polymyositis,Dermatomyositis, Spondyloarthropathies such as ankylosing spondylitis,or Sjogren's Syndrome.

The duration, dosages and frequency of administering compounds orcompositions of the invention may be adapted according to the subjectand disease. The treatment may be used alone or in combination withother active ingredients, either simultaneously or separately orsequentially.

The compounds or compositions according to the invention may beadministered in various ways or routes such as, without limitation, bysystemic injection, intramuscular, intravenous, intraperitoneal,cutaneous, subcutaneous, dermic, transdermic, intrathecal, ocular (forexample corneal) or rectal way, or by a topic administration on aninflammation site, and preferably by intramuscular or intravenousinjection.

A typical regimen comprises a single or repeated administration of aneffective amount of a GIBsPLA2 modulator over a period of one or severaldays, up to one year, and including between one week and about sixmonths. It is understood that the dosage of a pharmaceutical compound orcomposition of the invention administered in vivo will be dependent uponthe age, health, sex, and weight of the recipient (subject), kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe pharmaceutical effect desired. The ranges of effectives dosesprovided herein are not intended to be limiting and represent preferreddose ranges. However, the most preferred dosage will be tailored to theindividual subject, as is understood and determinable by one skilled inthe relevant arts (see, e.g., Berkowet et al., eds., The Merck Manual,16^(th) edition, Merck and Co., Rahway, N.J., 1992; Goodmanetna., eds.,Goodman and Cilman's The pharmacological Basis of Therapeutics, 10^(th)edition, Pergamon Press, Inc., Elmsford, N.Y., (2001)).

Diagnosis

The invention also provides methods for detecting an immune defect in asubject based on a detection of the presence or amount or absence ofGIBsPLA2 in a sample from a subject. The method of the invention may becarried out using a variety of detection technologies or platforms knownper se in the art such as, without limitation Capture assay, Sandwichassay, Competition assay, Radio-immuno assays, Enzyme labels withsubstrates that generate colored, fluorescent, chemiluminescent, orelectrochemically-active products, Fluorescence, fluorescentpolarization, Chemiluminescence, Optical and colorimetric,Electrochemiluminescence, Time-resolved fluorescence, Surface plasmonresonance, Evanescent wave, Multiwell plate (ELISA), Individual assay,Multiplex assay, Latex bead—multiplex assay, Microarray (Laminarsurface)—multiplex assay, Glass, Plate based assays or Strip basedassays.

In a particular embodiment, the method comprises determining thepresence, or amount, or absence of a polymorphism in the GIBsPLA2 gene,RNA or protein. Our results show that GIBsPLA2 is subject to highpolymorphism and that this correlates to the physiological status ofsubjects. The invention thus comprises (i) determining the presence, oramount, or absence of a particular polymorphic isoform of GIBsPLA2,and/or (ii) determining the global rate of polymorphism of GIBsPLA2 in asubject, said data being correlated to the physiological status of thesubject. In particular, specific isoforms may be characteristic of thepredisposition, presence or onset in a subject of a disorder asdescribed above. Such determination may also be used in personalizedmedicine, to adjust treatment.

Methods of Monitoring and/or Diagnosing Immunodeficiency Associated withCD4 T Cell Defects Comprising Detecting GIBsPLA2

Methods of monitoring and/or diagnosing immunodeficiency associated toCD4 T cell defects in particular in human immunodeficiency virus (HIV)infection in a subject, are provided by this disclosure. In someembodiments the methods comprise (a) providing a sample containing abody fluid, preferably plasma from a subject, and (b) detecting a levelof GIBsPLA2 in the sample above a threshold. The presence of GIBsPLA2 inthe sample may be detected by any method known in the art, such as forexample by a method comprising an enzymatic assay, a ligand-captureassay and/or an immunoassay.

In some embodiments the method comprises obtaining a sample comprisingplasma from a subject and determining whether the plasma has at leastone activity selected from inducing formation of abnormal membranemicrodomains (MMD) in CD4 T cells from healthy subjects and renderingCD4 T cells of healthy subject refractory to interleukin-7 (IL-7)signaling. If the plasma from the subject comprises such an activitythen the subject is in some embodiments determined to have a CD4 Tcell-linked immunodeficiency as often observed in HIV-infected patientsbut not only. If the plasma fraction does not comprise such an activitythen the subject is in some embodiments determined to have low exposureto immunodeficiency associated to the alteration of T CD4 cells tocytokine-regulated homeostasis.

In some embodiments the subject is determined to have an HIV infection.In contrast, if the protein fraction does not comprise such an activitythen the subject is in some embodiments determined to not have animmunodeficiency associated to CD4 T cell defects as disclosed herein.In some embodiments the subject is determined to not have an HIVinfection.

In some embodiments the methods comprise contacting the samplecomprising a body fluid, preferably plasma, from the subject with anantibody specific for GIBsPLA2 and determining the presence or absenceof an immunological reaction. In some embodiments the presence orabsence of an immunological reaction is determined by a methodcomprising an enzyme-linked immunosorbent assay (ELISA). The presence ofan immunological reaction between the antibody specific for GIBsPLA2 andthe sample indicates the presence of GIBsPLA2 in the sample, which inturn indicates that the subject has an immunodeficiency associated toCD4 T cell defects. In some embodiments the subject is determined tohave an HIV infection. In contrast, the absence of an immunologicalreaction between the antibody specific for GIBsPLA2 and the sampleindicates that the subject does not have an immunodeficiency associatedto CD4 T cell defects as disclosed herein. In some embodiments thesubject is determined to not have an HIV infection.

In some embodiments the assay for the presence of GIBsPLA2 in the sampleis qualitative. In some embodiments the assay for the presence ofGIBsPLA2 in the sample is quantitative.

In some embodiments the methods comprise comparing the results of theassay to the results of a similar assay of a control sample comprisingplasma of a subject who does not have an immunodeficiency associated toCD4 T cell defects. In some embodiments the methods comprise comparingthe results of the assay to the results of a similar assay of a samplecomprising plasma of the same subject harvested earlier.

Methods of Monitoring and/or Diagnosing Immunodeficiency Associated withCD4 T Cell Alteration Comprising Characterizing Membrane Microdomains onCD4 T Cells

The data in the examples demonstrate that HIV-infected patients presentformation of distinctive membrane microdomains (MMD) on the surface ofCD4 T cells although very few cells are really infected by HIV.Accordingly, this disclosure also provides methods for diagnosingimmunodeficiency associated with CD4 T cell alteration, such as forexample immunodeficiency caused by human immunodeficiency virus (HIV)infection in a subject. In some embodiments the methods comprise: (a)isolating CD4 T lymphocytes from a subject, and (b) measuring the numberand/or size of membrane microdomains (MMD) on the T-cells. In someembodiments the methods further comprise at least one of (c) measuringthe amount of phospho-STATS in the T-cells and (d) assaying the nuclearimport fraction of phospho-STATS in the T-cells. In some embodiments thenumber and/or size of MMD on the T-cells is measured in the absence ofinterleukin. In some embodiments the number and/or size of MMD on theT-cells is measured in the absence of IL-2. In some embodiments thenumber and/or size of MMD on the T-cells is measured in the absence ofIL-7. In some embodiments the number and/or size of MMD on the T-cellsis measured in the presence of a subthreshold level of interleukin.

In some embodiments if the number of MMD on the T cells isolated fromthe subject is at least a threshold that indicates that the subject hasimmunodeficiency associated with CD4 T cell alteration. In someembodiments it indicates that the subject has an HIV infection. In someembodiments if the number of MMD on the T cells isolated from thesubject is not at least a threshold that indicates that the subject doesnot have immunodeficiency associated with CD4 T cell alteration asdisclosed herein. In some embodiments it means that the subject does nothave an impaired CD-4 T cell response to cytokine signaling. In someembodiments it means that the subject does not have an impaired CD-4 Tcell response to interleukin-7. In some embodiments it indicates thatthe subject does not have an HIV infection. In some embodiments thethreshold is at least about 80 per cell, at least about 90 per cell, atleast about 100 per cell, at least about 110 per cell, or at least about120 per cell. In a non-limiting preferred embodiment, the threshold isat about 100 per cell. In some embodiments if the MMD on the T cellsisolated from the subject have a diameter of at least a threshold thatindicates that the subject has an HIV infection. In some embodiments ifthe MMD on the T cells isolated from the subject do not have diameter ofat least a threshold that indicates that the subject does not have animpaired response to interleukin-7 and more generally to cytokines. Insome embodiments it indicates that the subject does not have an HIVinfection. In some embodiments the threshold is a diameter of at least100 nm, at least 110 nm, at least 120 nm, at least 130 nm, or at least140 nm. In a non-limiting preferred embodiment, the threshold is adiameter of at least about 120 nm.

Because RIF may alter the responsiveness of CD4 T cells to IL-7 byaggregating membrane receptors in abnormally large MMD, responses toother gamma-c and cytokines may be affected as well and RIF might bealso associated to other pathologies involving altered CD4 T cellresponse.

Methods of Identifying Candidate Therapeutic Agents

This invention also provides methods for identifying a candidatetherapeutic agent, comprising: (a) contacting CD4 T lymphocytes withGIBsPLA2 in the presence of an agent, and (b) measuring GIBsPLA2-inducedCD4 T cell activation. In some embodiments the methods comprise (c)comparing the level of GIBsPLA2-induced CD4 T cell activation in thepresence of the agent with the level of GIBsPLA2-induced CD4 T cellactivation in the absence of the agent. In some embodiments, if thelevel of GIBsPLA2-induced CD4 T cell activation in the presence of theagent is lower than the level of GIBsPLA2-induced CD4 T cell activationin the absence of the agent, then the agent is identified as a candidateimmunodeficiency therapeutic agent. In some embodiments the agent isidentified as a candidate HIV therapeutic agent. In some embodiments, ifthe level of GIBsPLA2-induced CD4 T cell activation in the presence ofthe agent is higher than the level of GIBsPLA2-induced CD4 T cellactivation in the absence of the agent then the agent is identified as acandidate immunosuppressive therapeutic agent.

In some embodiments, measuring GIBsPLA2-induced CD4 T cell activationcomprises determining the number of MMD per CD4 T cell.

In some embodiments, measuring GIBsPLA2-induced CD4 T cell activationcomprises determining the mean diameter of MMD on CD4 T cells.

In some embodiments, measuring GIBsPLA2-induced CD4 T cell activationcomprises determining the IL-7 responsiveness of CD4 T cells assayed bySTATS phosphorylation and/or nuclear import.

As used herein an “agent” may be any chemical entity under evaluation asa potential therapeutic. In some embodiments the agent is an organicmolecule. In some embodiments the agent comprises from 2 to 100 carbonatoms, such as from 2 to 50 carbon atoms, 5 to 50 carbon atoms, or 10 to50 carbon atoms. In some embodiments the agent is a peptide, a protein,a glyco-protein, or a lipoprotein. In some embodiments the agent is anantibody.

In some embodiments the agent has not been previously determined to havea biological activity implying an utility as a therapeutic agent fortreatment of immunodeficiency, such as that often associated with HIVinfection. In some embodiments the agent has been previously determinedto have a biological activity implying an utility as a therapeutic agentfor treatment of immunodeficiency such as that often associated with HIVinfection.

As used herein, a “candidate immunodeficiency therapeutic agent” or a“candidate HIV therapeutic agent” is an agent that inhibits the abilityof RIF to activate CD4 T cells in at least one assay. Consistent withthe data reported herein, the ability of an agent to inhibit the abilityof GIBsPLA2 to activate CD4 T cells in at least one assay is a usefulway to identify agents that are likely to be therapeutically useful fortreating immunodeficiencies including immunodeficiencies associated withHIV infections. Accordingly, it is also a useful way to identify agentsthat are likely to be therapeutically useful for treating HIV infection.Of course, as with all therapeutic molecules further characterizationwill be required. However, this does not detract from the utility ofcandidate HIV therapeutic agents of this disclosure.

Further aspects and advantages of the invention are disclosed in thefollowing experimental section, which shall be considered asillustrative.

EXAMPLES

1. Materials and Methods

1.1. Patients

VP included in the study had been HIV-positive for more than one year.They had never received any antiretroviral drugs and had a viralload >10,000 RNA copies/ml with a CD4 count >200/μl at the time of bloodcollection (ANRS EP 33 and EP20 studies). All blood samples from VP weredrawn at the Centre Hospitalier de Gonesse. Blood from HD was providedby the Etablissement Francais du Sang (Centre Necker-Cabanel, Paris).Plasma samples from ART patients were drawn from individuals who hadbeen receiving treatment for at least one year. Their viral load hadbeen undetectable for at least 6 months and their CD4 counts >500/μl atthe time of blood collection. Plasma samples from HIC patients weredrawn from individuals with an undetectable viral load 10 years afterinfection. Plasma samples were collected at Centre d'InfectiologieNecker-Pasteur.

1.2. Analysis of Membrane Microdomains (MMD), Receptor Diffusion Ratesand Phospho-STATS Cellular Compartmentalization in Purified CD4 TLymphocytes

CD4 T-cells were purified by negative selection as already described(10) then activated with 2 nM recombinant glycosylated human IL-7(Cytheris) or 40 μg PHA (Sigma). The confocal and STED microscopy usedto study cell surface microdomains (MMD) and phospho-STATS cellularcompartment distribution has already been described (10, 12). FCSanalysis of protein diffusion at the surface of living cells has alsobeen described (10, 12).

1.3. Preparation and Analysis of Detergent-Resistant Microdomains (DRM)

The preparation of Triton-X100 lysates of CD4 T lymphocytes from HD orVP, followed by centrifugation through sucrose gradients and Westernblot analysis of the fractions collected, has been previously described(12). mAb specific for flotillin, IL-7Ralpha and gamma c were used todetect the corresponding bands by Western blots (12).

1.4. Characterization of RIF from VP Plasma

1.4.1. Bioassays

The MMD induction assay was as follows: VP plasma (5 or 10%) was firstincubated (20 min) in medium with purified HD CD4 T cells. The cellswere then plated on polylysine-coated glass slides for 10 min thenactivated by 15 min IL-7 (2 nM) or not for control (NS), then fixed byPFA (PFA, 1.5%, 15 min at 37° C. followed by 15 min at room temperature)equilibrated one hour in PBS/SVF 5% before being stained by choleratoxin B (CtxB-AF488). MMD were counted by STED microscopy.

The assay for inhibition of STAT phosphorylation and nucleartranslocation was as follows: VP plasma (5 or 10%) was first incubatedwith purified HD CD4 T cells (20 min) before stimulation by IL-7 (2 nM,15 min.). Cells were then plated on polylysine-coated glass slides for10 min then activated by 15 min IL-7 (2 nM) or not for control (NS),then fixed by PFA (PFA, 1.5%, 15 min at 37° C. followed by 15 min atroom temperature) and permeabilization by methanol (90% at −20° C.).Cells were equilibrated one hour in PBS/SVF 5% then phospho-STATS wasthen stained by rabbit anti-STATS labelled with goat anti-rabbit-Atto642and analyzed by FACS or STED microscopy.

1.4.2. Enzyme Treatments

The effects of enzyme digestion on RIF activity were evaluated bytreating VP plasma filtered on a 30 kDa membrane. Plasma compounds withMW<10 kDa were used as negative controls. Effects of porcine trypsin (1U/ml for 30 min at 37° C., followed by PMSF inhibition and bufferexchange with Millipore 5 kDa-membrane centrifugal filters), or DNAse I(1 U/ml for 30 min at 37° C.), or RNAse (1 U/ml for 30 min at 37° C.) orPeptide N-glycanase (1 U/ml for 30 min at 37° C.) were tested. Allpreparations were analyzed at 10% final concentration.

1.4.3. MW Determination or RIF Purification

Size exclusion chromatography was performed by loading 1.6 ml of plasmaonto a 85-ml Sephadex G100 column pre-equilibrated with ammoniumcarbonate (0.1M) or PBS, then collecting 0.8 ml fractions of the eluate.The column was calibrated using a protein set (GE-Healthcare). Proteinconcentration was measured by the Bradford method. VP plasma previouslyfiltered on a 100 kDa membrane and total VP plasma were tested and gaveidentical results. Fractions between 13-17 kDa were collected, whichcontain semi-purified RIF.

1.4.4 Isoelectric Point Determination

Anion or cation exchange chromatography was performed on MonoQ or MonoS1 ml columns (GE-Healthcare) with elution by successive pH steps(ammonium carbonate/ammonium acetate buffers). The pH of each eluatedfraction was measured and these were then adjusted to pH 7.4 beforetesting of their biological effects. RIF activity was measured in thecorresponding fractions immediately after elution.

1.4.5 MS Analysis

Samples from gel filtration (G100) were lyophilized then resuspended,pooled and proteolysed with porcine trypsin, according to methods knownper se in the art. Proteolytic peptides were then separated in 12fractions by chromatography through C18 column eluted in ammoniumacetate. The 12 fractions were separated through C18 eluted in reversephase (acetonitrile) and directly injected by electrospray in anorbitrap Velos (Thermo Scientific) for MS analysis with secondaryAr-fragmentation then MS/MS for the 10 higher-intensity peaks per MSscan.

Standard Mascot and X-Tandem programs were used. For each protein ofdatabase subsets, 3 criteria were computed:

-   -   i-score: Computation of theoretical specificity of every        peptides from trypsin digestion of a single protein in the        NextProt database enriched with mature proteins with signal        peptide cleavage (number of unique peptides/protein): number of        specific peptides overall human sequences (all), sequences with        a N-term signal peptide (sec) per protein    -   Computation of the theoretical occurrence of peptides compatible        with peaks from all MS scan series (theoretical peptide matching        peaks/protein)    -   Computation of the theoretical coverage of protein sequence with        peak-matching peptides

For each protein a p score was determined as a computation of all threescores.

Example 1: Aberrant Activation of CD4 T Lymphocytes from VP as Measuredby the Presence of Abnormal Membrane Microdomains (MMD)

This example describes the investigation of new molecular and cellularparameters that explain some of the abnormal responses seen in the CD4 Tlymphocytes of chronically HIV-infected patients. Chronic activation ofthe immune system is usually measured by assessing the over expressionof cell surface molecules such as CD38, HLA-DR and CD25 that areconsidered as the main markers of CD4 dysfunction (15). However, despitemany efforts, these data have remained blurred, and the phenotype of theCD4 T cells cannot directly explain their immune defects.

STED microscopy and labeling with cholera toxin B (CtxB-AF488) were usedto detect the presence of MMD (12). Before any stimulation, the surfaceof CD4 T lymphocytes purified from VP was found to bear far more MMDthan quiescent CD4 T lymphocytes purified from HD (FIG. 1A). And mostimportantly, all the CD4 T cells from VP showed increased numbers ofMMD. This abnormal pattern was not the consequence of stimulation byIL-7 in VP plasma since average IL-7 concentrations in this plasma (0.4pM) were only 100th the Kd of the IL-7R (13, 14). When purified CD4 Tcells from HD were stimulated by IL-7, large numbers of MMD wereobserved. By contrast, the MMD pattern of CD4 T cells from VP wasunaffected by IL-7 (FIG. 1A). This abnormal activation coupled with theabsence of any response to IL-7 can be mimicked by a non physiologicalstimulus such as with phytohemagglutinin (PHA) (FIG. 1A).

These various abnormal MMD were then counted. Around 150-200 MMD wereobserved per CD4 T cell from VP, as with PHA-stimulated HD CD4 T cells(FIG. 1C). Here again, the results obtained showed that all CD4 T cellsfrom VP expressed MMD, including all the major CD4 subpopulations (FIG.1C). IL-7 failed to increase MMD numbers in VP. By contrast, MMD numbersin HD CD4 T cells increased from a background level of around 10MMD/cell to 300 after IL-7 stimulation. A study of MMD size was alsoconducted (FIGS. 1D, 1E). This showed that the MMD on CD4 T cells fromVP and on PHA-stimulated HD CD4 T cells were far larger (250 nm) thanthose from HD CD4 T cells stimulated by IL-7 (90 nm).

Example 2: All IL-7R Alpha and Gamma-c Chains are Sequestered inAbnormal Detergent-Resistant Membrane Microdomains (DRM) Isolated fromVP CD4 T Cells

Resting HD CD4 T cells were analyzed to verify that IL-7R alpha andgamma-c chains are located in high-density fractions outside MMD. Whenthese HD CD4 T cells are stimulated by IL-7, these two chains arelocated in low-density fractions corresponding to detergent-resistantMMD or DRM containing all the proteins sequestered in MMD (FIGS. 2A-2C).

When the study was repeated on CD4 T cells purified from VP, the patternwas different (FIGS. 2A-2C). Before any stimulation, all the IL-7R alphaand gamma-c chains were already sequestered in DRM; none were located inthe high-density fractions corresponding to free receptors outside theMMD. Furthermore, pre-stimulation of the CD4 T cells by IL-7, before DRMpreparation, did not affect this pattern (data not shown). Here again,pre-stimulation of HD CD4 T cells by non physiological PHA reproducedthis pathological situation. This confirms the data in FIGS. 1A-1E anddemonstrates that the CD4 T cells in VP are subject to aberrantactivation prior to any stimulation. In addition, these abnormal MMDcontain all the IL-7R chains (FIGS. 2A-2C).

Example 3: 2D Gel Analysis of the IL-7 Signalosome in Purified CD4 TCells from HD, VP and IL-7-Stimulated HD Cells. Characterization of theAberrant State of Activation by the Protein Pattern Recovered afterImmunoprecipitation

2D-electrophoresis was used to demonstrate that the composition of theIL-7 signalosome in VP was abnormal and different from that in quiescentand IL-7-activated HD CD4 T cells (FIGS. 7A-7C).

Proteins were immunoprecipitated with anti-IL-7Ralpha (mouse mAb 40131,R&D System) immobilized on protein G-Sepharose 4G from purified CD4T-cell lysate and separated on 2D-PAGE (IEF on pH 3-10 gel stripesfollowed by SDS-gel with 12% acrylamide-bis). pH and MW (kDa) scales aredisplayed. Gels were stained with Sypro-Ruby. The gels shown arerepresentative of 8 NS/IL-7 pairs obtained from HD and 3 gels from VP.

(FIG. 7A) non-stimulated (NS) HD CD4 T-cells.

(FIG. 7B) VP CD4 T-cells. More spots were observed in Sypro Ruby-stained2D-gels prepared from VP than from HD. In addition we observed thatcommon spots were more intense when 2D-gels were prepared with VPextracts.

(FIG. 7C) IL-7-stimulated HD CD4 T-cells. The pattern in HD CD4 T cellsstimulated by IL-7 differs from that in VP CD4 T cells. This furthersupports the proposal that the aberrant activation found in VP is notthe consequence of IL-7 stimulation that could take place in organs withhigh levels of IL-7, for example in IL-7-producing organs.

It may be concluded from this analysis that IL-7R chains in VP CD4 Tcells are not only part of abnormal MMD but also that they interact withprotein complexes different from those found in the normal IL-7signalosome.

Example 4: Diffusion Rate of IL-7Ralpha at the Surface of Purified CD4 TCells from HD, VP and PHA-Stimulated HD Cells. IL-7Ralpha in VP CD4 TCells is Embedded in Lipid-Rich Abnormal MMD, Thus Limiting itsDiffusion Rates and Precluding any Interactions with the Cytoskeletonand Therefore any Ability to Transmit Signals

The two-dimensional diffusion of IL-7Ralpha stained withAF488-anti-IL-7Ralpha mAb was measured by FCS at the surface of livingCD4 T-cells. The results are shown in FIGS. 8A-8G. Diffusion times τD(in 10⁻³ sec) were measured in the absence of IL-7 (◯, autocorrelation)or in the presence of IL-7-biotin SAF633 (●, crosscorrelation) asdescribed (10, 12). These times were then plotted versus cell surfacearea ω₀ ² (in 10³ nm²) intercepted by the confocal volume. The diffusionplots are shown with and without pre-treatment with MMD inhibitors(COase 1 μg/ml plus SMase 0.1 μg/ml for 30 min) or cytoskeletoninhibitors (CytD 20 μM plus Col 10 μM for 30 min).

Bars indicate SEM from 5 independent experiments. Slopes of the linearregression give effective diffusion rates D_(eff) and y-interceptsextrapolate confinement time τ0 as we described previously (12). D_(eff)are shown in the bar graph FIG. 3A.

(FIGS. 8A, 8D) at the surface of HD CD4 T-cells,

(FIGS. 8B, 8E) at the surface of VP CD4 T cells,

(FIGS. 8C, 8F) at the surface of HD CD4 T cells pre-activated with PHA(1 μg/ml).

(FIG. 8G) Scheme of the mechanism of IL-7Ralpha diffusion embedded inMMD before and after treatment by MMD inhibitors or cytoskeletoninhibitors. MMD are indicated by disks, receptors by rods, cytoskeletonis shown as a net. Diffusion rates (fast, slow, very slow) are indicatedto facilitate data interpretation. This scheme illustrates the resultsalso reported in FIG. 3A.

Example 5: IL-7R Chains Sequestered in the Abnormal MMD of VP CD4 TCells are Non Functional

IL-7R alpha diffusion rates were measured at the surface of CD4 T cellsas previously described (10, 12) and as detailed in Example 4. Beforeany stimulation, these diffusion rates were seen to be three timesslower on VP than HD CD4 T cells (FIG. 3A). This further demonstratesthat IL-7R alpha chains are embedded in abnormal MMD at the surface ofthese CD4 T cells (FIG. 3A). COase plus SMase treatment released thereceptor from its MMD constraints and therefore increased its diffusionrate (FIG. 3A). By contrast, treatment with cytochalasin D (Cyt D) pluscolchicine (Col)—which disorganizes the cytoskeleton—had no effect onthe diffusion rate of the IL-7R alpha chain in VP CD4 T cells (FIG. 3A).Since cytoskeleton organization is an absolute necessity for signaltransduction, this absence of any functional or structural link betweenIL-7R alpha and the cytoskeleton meshwork suggests that signaling cannotproceed when IL-7R complexes are sequestered in abnormal MMD, as is thecase in VP CD4 T cells.

Pulsed-STED microscopy was then used to study STATS phosphorylation(phospho-STATS) and phospho-STATS partition in the cytoplasm and nucleusof both HD and VP CD4 T cells. FIG. 3B shows STED images ofphospho-STATS distribution before and after 15 min of IL-7 stimulation.We noted that phospho-STATS accumulated in the nucleus of HD CD4 Tcells, and this phenomenon was inhibited by cytoskeletondisorganization. By contrast, no phospho-STATS translocation to thenucleus occurred in VP CD4 T cells or in PHA pre-stimulated HD CD4 Tcells (FIG. 3B).

The kinetics of phospho-STATS appearance in the cytoplasm and nucleuswas then followed for one hour (FIGS. 3C, 3D, 3E). This showed thatphospho-STATS in VP CD4 T cells mostly accumulated in the cytoplasm anddid not migrate to the nucleus (FIG. 3D), as in PHA-stimulated HD CD4 Tcells (FIG. 3E). This was particularly clear when the results werecompared with those obtained in the five minutes following IL-7stimulation of HD CD4 T cells where 50% of phospho-STATS was found inthe nucleus (FIG. 3C).

Example 6: Plasma from VP Induces Abnormal MMD at the Surface ofPurified HD CD4 T Cells

The origin of the aberrant activation of VP CD4 T cells was theninvestigated. The fact that all the CD4 T cells were involved and that anon physiological signal such as PHA mimics the results led to aninvestigation of the plasma of VP. Purified HD CD4 T cells wereincubated with 10% VP plasma for 30 min and MMD counted at the surfaceof the CD4 T cells as detected by labeled cholera toxin B (CtxB-AF488).FIG. 4A shows the images obtained. VP plasma alone induced large numbersof MMD on HD CD4 T cells. Adding IL-7 did not affect the size or numberof these MMD (FIG. 4A). These results are shown for plasma from fivedifferent VP (FIG. 4B) and were verified using many more plasma samplesfrom these VP (>15). The experiments were also repeated using CD4 Tcells from different HD (>5). Controls consisted of testing plasmasamples from HIV-controllers (HIC) and antiretroviral-treated (ART)patients on purified HD CD4 T cells. None of these induced MMD orinhibited the IL-7 induction of MMD (FIG. 4C).

This was further verified by testing a large number of dilutions of thevarious plasmas (FIG. 4D). VP plasma down to a 0.1% dilution resulted inthe formation of MMD scattered across the cell surface. VP plasmadiluted 50 to 100 fold gave 50% maximum activity. None of the plasmasamples from HIC or ART patients induced MMD at any dilution.

Example 7: Plasma from VP Inhibits IL-7-Induced Phospho-STATS NuclearTranslocation

The function of the IL-7R in HD CD4 T cells treated with VP plasma wastested by following STATS phosphorylation and nuclear translocation. Asseen in FIG. 5A, pre-incubation of HD CD4 T cells with VP plasma (10%concentration) inhibited IL-7-induced STATS phosphorylation and itsnuclear translocation. FIG. 5B shows the results obtained with five VPplasma samples. All at a 10% dilution inhibited the nucleartranslocation of phospho-STATS. These results were confirmed withplasmas from different VP (>15) and various sources of HD CD4 T cells(>5).

The effect of plasma derived from HIC and ART patients was also testedby pre-incubating these with purified HD CD4 T cells (FIGS. 5A, 5C).Here again, only VP plasma was able to inhibit the IL-7-induced nucleartranslocation of phospho-STATS. It was also determined (FIG. 5D) that VPplasma was active down to a 0.1% dilution, and half maximum activity wasobtained at a 50 to 100 fold dilution, thus correlating with the abilityto induce abnormal MMD (FIG. 4D).

The effect of plasma derived from ART-treated patients butnon-responsive (CD4-NR) to their treatment (low count of viral RNA andlow count of CD4 T-cells) was also tested by pre-incubating these withpurified HD CD4 T cells. Here again, only CD4-NR plasma was able toinhibit the IL-7-induced nuclear translocation of phospho-STATS. It wasalso determined that CD4-NR plasma was active down to a 0.1% dilution,and half maximum activity was obtained at a 50 to 100 fold dilution,thus correlating with the ability to induce abnormal MMD as observedwith VP.

Example 8: Molecular Characterization of the Refractory State InducingFactor

The chemical nature of RIF was investigated. The studies performed (FIG.6A) showed that RIF is a protein since its activity was destroyed bytrypsin. Treatment with peptide N-glycanase (PNGase) had no effect,indicating that N-glycosylation is not required for RIF activity.

The molecular weight of RIF was then measured by size-exclusionchromatography on Sephadex G-100. Induction of MMD (FIG. 6B) andinhibition of IL-7-induced phospho-STATS nuclear translocation (FIG. 6C)was measured for all fractions eluted from the column. Tworepresentative column profiles are given in FIGS. 6B and 6C. Both showthat RIF is a single factor with a MW between 10 and 15 kDa.

FIG. 6B shows the densities of the viral peptides or proteins measuredby dot blot in each of the 100 fractions collected from the SephadexG100 column. Measurements were repeated three times with differentpolyclonal antibodies from VP plasma samples characterized by their highactivity against viral proteins. For each experiment the signalsobtained with HD plasmas were then subtracted from the values. Thepattern shown in FIG. 6B demonstrates that no viral proteins orfragments were detected in the fraction containing RIF activity whilethe dot blot assay was able to detect viral proteins at higher MW (from190 to 32 kDa).

Ten to 15 kDa active, enriched fractions from the Sephadex G100 columnswere then used to frame the isoelectric point of RIF by retention onanion (MonoQ) or cation (MonoS) exchange columns followed by pH elution(pH increase with MonoS or pH decrease with MonoQ) (FIG. 6D). TheMMD-inducing activity of the various pH fractions was then measuredafter adjusting their pH to 7.4. In all, 25 to 30% of the initialactivity was recovered in two fractions, a result consistent with anisoelectric point of 6.5 to 8.0.

RIF is therefore a secreted protein, with a MW of about 15 kDa, a pIaround 7.5-8.0, which contains disulfide bridge. Following the abovestructural and functional features, RIF identity was directly obtained.In particular, amongst all of the 36853 known human proteins, 62 onlyhad the above four characteristics of RIF. Semi-purified materialprepared from three viremic patients and three HD were analyzed usingmass spectrometry and standard Mascot program. Proteins recovered wereranked according to the p score described in Materials and Methods. Theresults shown in Table 1 below clearly and directly indicate that RIF isGIBsPLA2.

TABLE 1 Mnemonic ID PI MW i_s p_score description PA21B_HUMAN P040547.95 14138.99 9 0.64 phospholipase A2 group 1 TMEM9_HUMAN Q9P0T7 6.2318568.37 5 0.29 Transmembrane protein 9 (TM) ESM1_HUMAN Q9NQ30 6.8318122.42 5 0.10 Endothelial cell-spe molecule 1 CYTD_HUMAN P28325 6.7613858.6 3 0.08 Cystatin-D SSRB_HUMAN P43308 7.03 18273.74 7 0.05 Signalseq R sub beta (TM) GPIX_HUMAN P14770 6.14 17316.06 6 0.04 Plateletglycoprotein IX B2MG_HUMAN P61769 7.67 18510.47 4 0.03Beta-2-microglobulin EPGN_HUMAN Q6UW09 7.72 14724.99 1 0.02 EpigenIL19_HUMAN Q9UHD0 7.8 17812.56 5 0.02 Interleukin-19 IL3_HUMAN P087007.05 15091.38 3 0.02 Interleukin-3 GML_HUMAN Q99445 6.67 15918.41 7 0.02Glycosyl-PPI-anc like protein CYTM_HUMAN P05113 7.02 13149.22 4 0.017Cystatin-M

The protein found in the plasma of viremic patients is thus the secretedform of GIBsPLA2. The mature protein has 125 aa (MW14138), PI 7.95 and 7disulfide bridges. Using commercial purified porcine GIBsPLA2, we wereable to verify in vitro that this protein induces abnormal MDM, whichblock IL-7 pSTAT5 nuclear translocation in the plasma of viremicpatients, further confirming that RIF is GIBsPLA2, more specifically thesecreted form thereof. The amino acid sequence of a human GIBsPLA2 isprovided as SEQ ID NO: 2.

Example 9: PLA2sGIB Induces Unresponsiveness (Anergy) of CD4 Lymphocytes

Example 7 shows that PLA2sGIB, through induction of aMMD, induces ablockade of IL-7-induced nuclear translocation of phospho STATS (NTpSTAT5). Consequently, CD4 T lymphocytes do not respond to IL-7 anddespite of the high level of this cytokine in the plasma of HIVpatients, their number decreases then leading to CD4 lymphopenia thehallmark of HIV-infected patients.

Here we investigated the possibility that PLA2sGIB also participates tothe induction of anergy, another characteristic of the CD4 lymphocytesfrom chronically HIV-infected patients.

Bioassay

MMD Induction:

VP plasma containing PLA2sGIB was first incubated (20 min) in mediumwith purified HD CD4 T cells. The cells were then plated onpolylysine-coated glass slides for an additional 10 min. They were thenfixed with paraformaldehyde (PFA, 1.5%, 15 min at 37° C. followed by 15min at room temperature) before being stained by cholera toxin B(CtxB-AF488), MMD were counted by CW-STED microscopy.

Inhibition of STAT Phosphorylation and Nuclear Translocation:

VP plasma containing PLA2sGIB was first incubated with purified HD CD4 Tcells (20 min) before stimulation by IL-7 (2 nM, 15 min.). Cells werethen plated on polylysine coated glass slides before fixation by PFA(1.5%) and permeabilization by methanol (90% at −20° C.). pSTAT5 wasthen stained by rabbit anti-STATS labelled with goat anti-rabbit-Atto642and analyzed by FACS or pulsed STED microscopy.

Results

FIG. 10A shows that after exposition to PLA2 GIB (plasma of viremicpatient), CD4 lymphocytes from healthy donors (HD) become unable torespond to IL-2, as measured by the inhibition of the IL-2-induced NTpSTAT5. This inhibition is total with 3% plasma, and highly significantwith 1% plasma (p<0.0001).

We further studied the response of CD4⁺CD25⁺ T reg lymphocytes to PLA2GIB. The results are presented in FIG. 10B. As illustrated, while 100%of healthy cells respond to IL-2 by NT pSTAT5, PLA2 GIB (1% plasma ofviremic patients) completely inhibited this signal transductionmechanism. Since CD4⁺ CD25⁺ cells represent less than 5% of total CD4 Tcells, they cannot significantly influence the data presented in FIG.10A.

IL-7 and IL-2 are members of the gamma c cytokine family. To confirmthat unresponsiveness to this cytokine may be linked to gamma c, wetested the response to IL-4. IL-4 response was measured by following theIL-4 induced NT of pSTAT6 (FIG. 11 ). Our results clearly show that IL-4response is inhibited by PLA2 GIB (completely with 3% plasma and greatlywith 1% plasma).

These results therefore show that the signaling mechanisms induced bycytokines of the gamma c family are altered by PLA2 GIB. This is incomplete agreement with our finding that gamma c receptor chain is foundcompletely sequestered in aMMD spontaneously found at the surface of CD4lymphocytes from HIV-patients (data not shown).

Example 10: Activity of Recombinant Forms of PLA2 GIB

In this example, the activity of various purified forms of PLA2 GIBproteins was tested, to further confirm the effect of this protein inpurified form on the immune system, and to further confirm itsspecificity.

Enzymatic Assay

The assay was performed with the Enz Check PLA2 assay kit from LifeTechnologies (ref.: E102147). This assay provides a continuous rapidreal-time monitoring of PLA2 enzyme activities. The PLA2 activity isfollowed by the intensity increase of a single wavelength at 515 nm.PLA2 is detected by changes in the emission intensity ratio at 515/575nm with excitation at 460 nm. Specific activities are expressed inamount of fluorescent substrate (U) obtained per second and per μg ofenzyme in solution.

Results

The results are provided in Table 2 below.

TABLE 2 Activity of recombinant PLA2 GIB proteins Initial Final Specificconcentration concentration Quantity activity PLA2 Nature (mg/ml)(ug/ml) (ug) (U/ug/s) ppPLA2 IB Purified porcine 2.90 0.58 0.06 7694.31pancreas pPLA2 IB recombinant 1.40 2.80 0.14 10353.57 porcine (in E.coli) hPLA2 IB recombinant human 0.70 1.40 0.07 10694.57 (in E.coli)hPLA2 IIA recombinant human 1.45 2.90 0.15 214.93 (in E. coli) hPLA2 IIDrecombinant human 0.70 1.40 0.07 445.21 (E. coli) hPLA2 X recombinanthuman 0.68 1.36 0.07 3318.97 (in E. coli)

The results show that recombinant human PLA2 GIB produced in E. Coliexhibit a potent enzymatic activity. Furthermore, the results also showthat recombinant porcine PLA2GIB produced in E. Coli has a specificactivity similar to that of recombinant human PLA2GIB. By contrast,recombinant PLA2GIIA and PLA2GIID are not active and PLA2GX has a verylimited activity.

Recombinant PLA2 GIB thus represents a potent active agent for use inthe present invention.

Example 11: The Effects of PLA2sGIB on CD4 Lymphocytes Involve itsEnzymatic Activity

In this example, we investigated whether the activity of PLA2sGIB on CD4lymphocytes involved (e.g., was a consequence of) an enzymatic (e.g.,catalytic) activity of PLA2sGIB. Such enzymatic activity would modifythe membrane structure leading to the formation of multiple aMMD at thesurface of CD4 lymphocytes.

In these experiments, we tested a mutant of PLA2sGIB where a criticalhistidine at position 48 was replaced by glutamine (mutant H48Q). Usingthe enzymatic test described in example 10, we compared the enzymaticactivity of recombinant porcine PLA2 GIB produced in E. Coli with theactivity of mutant H48Q also produced in E. Coli. Each protein was usedat 200 microM. As shown FIG. 12 , the mutant has lost all of itsenzymatic activity, illustrating the critical role of histidine atposition 48 in PLA2 GIB.

We then compared the activity of wild type porcine PLA2 GIB with itsmutant H48Q in a bioassay. The results presented in FIGS. 13A-13B showthat the mutant has lost the ability of wtPLA2 GIB to induce aMMD or toreduce or abrogate IL-7 induced Nuclear Translocation of pSTAT5 (NTpSTAT5).

These results thus demonstrate that the enzymatic activity is involvedin the pathogenic effects of PL2 GIB on CD4 lymphocytes.

Example 12: Anti-GIBsPLA2 Antibodies Restore CD4-T Cell Activity in thePlasma of HIV Viremic Patients

This example illustrates that, in the plasma of viremic patients,GIBsPLA2 transforms CD4 lymphocytes from HD into “sick” lymphocytescomparable to those characterized in the blood of HIV-infected patients.This example further shows that anti-GIBsPLA2 antibodies do effectivelysuppress the pathogenic activity.

In a first series of experiments, the plasma were treated by sepharosebeads coated either by goat antibodies directed against human GIBsPLA2or by two control goat antibodies directed against non relevantantigens. FIG. 14A clearly shows that anti-GIBsPLA2 antibodiescompletely abolished or removed the activity of the plasma, which becameunable to induce abnormal MMD in CD4 lymphocytes from HD. Control I andcontrol II antibodies had no effect. These experiments were repeatedthree times for each plasma and three different plasma from viremicpatients were studied.

FIG. 14B shows identical results. Here the plasma were treated as abovebut were analyzed using the second bioassay. The plasma treated bysepharose beads coated with anti-GIBsPLA2 antibodies do not inhibitanymore IL-7-induced pSTAT5 nuclear translocation. Control I and controlII goats antibodies did not affect the ability of the plasma fromviremic patients to inhibit IL-7 induced pSTAT5 nuclear translocation.

In a second series of experiments, we tested the effects of neutralizingrabbit antibodies specifically directed against human GIBsPLA2, -GIIAand -GIID. These antibodies were incubated with the plasma and the cellsduring the bio assays. The results obtained show that anti-GIBsPLA2antibodies neutralize the effects of the viremic plasma as measured bythe induction of abnormal MMD and by inhibition of IL-7-induced pSTAT5nuclear translocation. It is noteworthy that antibodies directed againstsecreted PLA2-GIIA or secreted PLA2-GIID, two phospholipases which areclosely related to GIBsPLA2, had no effect in this test.

These results show that anti-GIBsPLA2 antibodies can revert and preventthe immunosuppressive effect of viremic plasma. These results show thatanti-GIBsPLA2 antibodies can prevent immunodeficiency and restimulatethe immune response in immuno-defective subjects.

These results further demonstrate that the response is specific.GIBsPLA2 is the only effector involved in the pathogenic effect examinedand characterizing the plasma of viremic patients.

Example 13: Anti-PLA2GIB Antibodies Inhibit PLA2 GIB Effects on CD4Cells

Cloned and purified human PLA2GIB was used to immunize rabbits.Immunoglobulin fractions of the corresponding sera were prepared. Theircapacity to inhibit the enzymatic activity of PLA2GIB was measured onradiolabelled E. Coli membranes. Active immunoglobulin fractions wereadded to the bioassay including CD4 Lymphocytes purified from the bloodof healthy donors. Cloned and purified secreted PLA2 (GIB, GIIA, GIIDand GX) were subsequently added to the cultures. As controlsimmunoglobulin fractions prepared from rabbits immunized with varioussecreted PLA2 were used.

FIG. 15 shows that different concentrations of polyclonal antibodyinhibit the induction of aMMD (upper graph) and block the IL-7-inducedNT pSTAT5 (lower graph). This activity can be obtained from 1 μg/ml to100 μg/ml of Ig containing anti-PLA2 GIB antibodies. This activity istotally specific since antibodies directed against PLA2 GIIA, PLA2GIIDor PLA2GX showed no effect in the bioassay.

These results thus further demonstrate that inhibiting PLA2GIB can beused to treat immunodeficiencies and to restore CD4 activity.

Example 14: Soluble PLA2GIB Receptor Inhibits PLA2 GIB Effects on CD4 TCells

As a further demonstration that inhibitors of PLA2GIB can exerttherapeutic effect, we tested a soluble form of a PLA2GIB receptor.

In a first series of experiment, we used, the soluble murine receptorspecific for PLA2 GIB having the following amino acid sequence (SEQ IDNO: 22):

MVQWLAMLQLLWLQQLLLLGIHQGIAQDLTHIQEPSLEWRDKGIEIIQSESLKTCIQAGKSVLTLENCKQPNEHMLWKWVSDDHLFNVGGSGCLGLNISALEQPLKLYECDSTLISLRWHCDRKMIEGPLQYKVQVKSDNTVVARKQIHRWIAYTSSGGDICEHPSRDLYTLKGNAHGMPCVFPFQFKGHWHHDCIREGQKEHLLWCATTSRYEEDEKWGFCPDPTSMKVFCDATWQRNGSSRICYQFNLLSSLSWNQAHSSCLMQGGALLSIADEDEEDFIRKHLSKVVKEVWIGLNQLDEKAGWQWSDGTPLSYLNWSQEITPGPFVEHHCGTLEVVSAAWRSRDCESTLPYICKRDLNHTAQGILEKDSWKYHATHCDPDWTPFNRKCYKLKKDRKSWLGALHSCQSNDSVLMDVASLAEVEFLVSLLRDENASETWIGLSSNKIPVSFEWSSGSSVIFTNWYPLEPRILPNRRQLCVSAEESDGRWKVKDCKERLFYICKKAGQVPADEQSGCPAGWERHGRFCYKIDTVLRSFEEASSGYYCSPALLTITSRFEQAFITSLISSVAEKDSYFWIALQDQNNTGEYTWKTVGQREPVQYTYWNTRQPSNRGGCVVVRGGSSLGRWEVKDCSDFKAMSLCKTPVKIWEKTELEERWPFHPCYMDWESATGLASCFKVFHSEKVLMKRSWREAEAFCEEFGAHLASFAHIEEENFVNELLHSKFNWTQERQFWIGFNRRNPLNAGSWAWSDGSPVVSSFLDNAYFEEDAKNCAVYKANKTLLPSNCASKHEWICRIPRDVRPKFPDWYQYDAPWLFYQNAEYLFHTHPAEWATFEFVCGWLRSDFLTIYSAQEQEFIHSKIKGLTKYGVKWWIGLEEGGARDQIQWSNGSPVIFQNWDKGREERVDSQRKRCVFISSITGLWGTENCSVPLPSICKRVKIWVIEKEKPPTQPGTCPKGWLYFNYKCFLVTIPKDPRELKTWTGAQEFCVAKGGTLVSIKSELEQAFITMNLFGQTTNVWIGLQSTNHEKWVNGKPLVYSNWSPSDIINIPSYNTTEFQKHIPLCALMSSNPNFHFTGKWYFDDCGKEGYGFVCEKMQDTLEHHVNVSDTSAIPSTLEYGNRTYKIIRGNMTWYAAGKSCRMHRAELASIPDAFHQAFLTVLLSRLGHTHWIGLSTTDNGQTFDWSDGTKSPFTYWKDEESAFLGDCAFADTNGRWHSTACESFLQGAICHVVTETKAFEHPGLCSETSVPWIKFKGNCYSFSTVLDSRSFEDAHEFCKSEGSNLLAIRDAAENSFLLEELLAFGSSVQMVWLNAQFDNNNKTLRWFDGTPTEQSNWGLRKPDMDHLKPHPCVVLRIPEGIWHFTPCEDKKGFICKMEAGIPAVTAQPEKGLSHSIVPVTVTLTLIIALGIFMLCFWIYKQKSDIFQRLTGSRGSYYPTLNFSTAHLEENILISDLEKNTNDEEVRDAPATESKRGHKGRPICISP

The inhibitor was tested in the bioassay described in example 9, at aconcentration of 100 nM. The results are presented in FIG. 16 . Theyshow that a recombinant PLA2 soluble receptor can be used as a potentantagonist and that such molecule is able to significantly block thenegative effect of PLA2sGIB on the NT of pSTAT5 (FIG. 16 ).

Similar results can be obtained in further sets of experiments usingPLA2-GIB-binding polypeptides comprising the sequence of SEQ ID NO: 25or 28.

Example 15: Overexpression of GIBsPLA2 Induces Immunological Deficiency

It has been previously shown that Highly Active Anti-Retroviral Therapy(HAART) which reduced viral load also induces a CD4 count increase inmost patients. However, in some patients, despite the fact that HIVbecomes undetectable, the CD4 counts do not increase. We have previouslystudied this clinical situation and we have shown that in these patientscalled CD4 Non Responders (CD4-NR) a strong and persistent defects ofthe CD4 T lymphocytes population is found.

FIGS. 17A-17B show that the plasma of CD4-NR patients do contains morePLA2 GIB activity than plasma from a viremic patient taken as control.This was first measured by the induction of abnormal MMD per cells.These data were also confirmed by measuring the ability to inhibitIL-7-induced pSTAT5 nuclear translocation.

Altogether, the results show that the plasma of CD4-NR patients containshundred times more PLA2 GIB activity than the plasma from viremicpatients.

Discussion

Our results show that PLA2 GIB induces an immunosuppression similar tothat which characterizes CD4 T cells from viremic patients, includingthe inability to respond to IL-2 (anergy) and to IL-7 (central mechanismtowards CD4 lymphopenia). Therefore, expression of GIBsPLA2 during HIVinfection plays a central role in the pathophysiology of the immunedisease that characterizes these patients. These defects are cell-typespecific since CD8 T lymphocytes from HIV patients do not exhibitabnormal MMD and continue to respond to IL-7 (data not shown). The modeof action of PLA2 GIB is probably the consequence of its enzymaticactivity. By attacking the membrane of CD4 lymphocyte, it modifies itsfluidity and probably allows the formation of abnormal and very largeMMD.

Inflammatory reactions play an important role during HIV infection.However, their exact role in HIV pathogenesis remains to be elucidated.Taking into account our data, one can hypothesize that HIV infectioninduces a very peculiar type of inflammation which includes GIBsPLA2.Furthermore, one can speculate that after PLA2 GIB induction, itssecretion escape to normal regulatory processes therefore leading to achronic production and to the immunological disorders which are thedirect consequence of the CD4 T lymphocytes dysfunction. As an indirectconsequence of the CD4 T lymphocytes dysfunction, other defects can alsobe observed. For instance, diminished production of Interferon gammawill decrease the functions of monocytes/macrophages and of naturalkillers.

Correlation between the recovery of plasmatic PLA2 GIB activity and thecharacteristics of different groups of patients is also veryinformative. “HIV controllers” are very rare patients which maintain anundetectable viral load and quasi normal CD4 counts over the years. Ourresults show that they do not express PLA2 GIB activity in their plasma.By contrast, in most patients, this enzyme is expressed and representsthe negative side of the inflammation which leads to the immunologicaldisease. Altogether, this clearly establishes that PLA2 GIB is a verycritical parameter in the pathophysiology of HIV infection.

HAART viral load decrease is followed by an immune restoration includingCD4 counts increase. During this treatment, PLA2 GIB activity disappearsin the plasma of the patients. Since, HAART is considered to decreasethe inflammatory reactions this further suggests that PLA2 GIB is partof these inflammatory processes. More importantly, we describe here thecase of the CD4-NR patients which remain with very low CD4 counts whileHAART control their viral load. The overproduction of PLA2 GIB found inthese individuals may explain the persistence of the immune disease thatcharacterizes this clinical status. Therefore, after HAART, there is astrong correlation between the decrease production of PLA2 GIB leadingto immune restoration or its persistent overproduction leading to theirreversibility of the immune disease.

The therapeutic consequences and utilities of this discovery are huge.Inhibition of PLA2 GIB may indeed be used to prevent or cure theimmunological disease of HIV patients as well as, more generally, ofimmunodepressed subjects. Applied early during infection, inhibitors ofPLA2 GIB lead the patients toward a HIV controller status. Appliedlater, alone or in conjunction/alternance with HAART, they acceleratethe recovery of the CD4 T lymphocytes functions and by boosting hostdefenses, inhibitors of PLA2 GIB lead to an equilibrium between thevirus and the immune system like in many other viral chronic infection.Therefore, inhibitors of PLA2 GIB represent very potent agents for use,alone or in combination, to treat disorders associated with an abnormalimmune response or activity. They can also help in sparing HAART andcould lead to the interruption of these treatments which are known fortheir severe detrimental effects.

Furthermore, since a lack of GIBsPLA2 expression (as in mice KO for thecorresponding gene) is well tolerated, transient or permanentsuppression of GIBsPLA2 using inhibitors or through vaccination,represents a strong and valid immunotherapy of immune diseases,particularly HIV patients.

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1. (canceled)
 2. A method for stimulating an immune response in aHIV-infected subject, or for treating AIDS in a HIV-infected subject,comprising administering to the subject an effective amount of ananti-GIBsPLA2 antibody or a fragment thereof that binds to secretedphospholipase A2 group IB (GIBsPLA2), wherein the HIV-infected subjectis a CD4 non-responder (CD4-NR).
 3. The method of claim 2, wherein theHIV-infected subject has low CD4 counts after prolonged ART.
 4. Themethod of claim 2, wherein said method increases CD4 counts in theHIV-infected subject having low CD4 counts after prolonged ART.
 5. Themethod of claim 2, wherein said method induces or stimulates CD4 T cellsactivation in the subject.
 6. The method of claim 2, wherein said methodsuppresses or reverses HIV-mediated immunodeficiency.
 7. The method ofclaim 2, wherein the anti-GIBsPLA2 antibody is selected fromanti-GIBsPLA2 polyclonal antibodies, anti-GIBsPLA2 monoclonal antibodiesor GIBsPLA2-binding fragments thereof selected from F(ab′)2 and Fabfragments, single-chain variable fragments (scFvs), single-domainantibody fragments (VHHs or Nanobodies) or bivalent antibody fragments(diabodies).
 8. The method of claim 2, wherein the anti-GIBsPLA2antibody or a fragment thereof, is human or humanized.
 9. The method ofclaim 2, wherein GIBsPLA2 is a protein comprising amino acid residues23-148 of SEQ ID NO: 2 or a naturally-occurring variant thereof.
 10. Themethod of claim 2, wherein the anti-GIBsPLA2 antibody or a fragmentthereof inhibits the expression or activity of GIBsPLA2.
 11. The methodof claim 10, wherein the activity of GIBsPLA2 is selected from inductionof formation of membrane microdomains (MMD) in CD4 T cells or renderingCD4 T cells refractory to interleukin signaling.
 12. The method of claim2, wherein the anti-GIBsPLA2 antibody is administered intramuscularly,subcutaneously, transdermally, intravenously, intraarterially, nasally,orally, mucosally, rectally or by inhalation.